Methods and compositions for treating cancer

ABSTRACT

Provided herein, in some embodiments, are methods and compositions (e.g., cell compositions) for the treatment of cancer.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/670,417 filed May 11, 2018; U.S. ProvisionalPatent Application Ser. No. 62/701,340 filed Jul. 20, 2018; U.S.Provisional Patent Application Ser. No. 62/756,643 filed Nov. 7, 2018;U.S. Provisional Patent Application Ser. No. 62/773,658 filed Nov. 30,2018; and U.S. Provisional Patent Application Ser. No. 62/826,600 filedMar. 29, 2019. The entire contents of the above-referenced patentapplications are incorporated herein by this reference.

BACKGROUND

Chimeric antigen receptor (CAR) T-cell therapy uses genetically-modifiedT cells to more specifically and efficiently target and kill cancercells. After T cells have been collected from the blood, the cells areengineered to include CARs on their surface. The CARs may be introducedinto the T cells using CRISPR/Cas9 gene editing technology. When theseallogeneic CAR T cells are injected into a patient, the receptors enablethe T cells to kill cancer cells.

SUMMARY

In some aspects, the present disclosure provides engineered immune cells(e.g., T cells) and methods of producing immune cells that have beenedited using CRISPR/Cas9 gene editing technology to disrupt endogenousCD70 expression (knockout CD70).

In some aspects of the present disclosure provide an engineered immunecell (e.g., T cell) comprising a disruption in the CD70 gene. In someembodiments, the engineered immune cells are allogeneic T cellscomprising a disrupted CD70 gene and a nucleic acid encoding a CAR. Insome embodiments, the engineered immune cells are allogeneic T cellscomprising a TRAC gene disrupted by insertion of a nucleic acid encodinga CAR, a disrupted β2M gene, and a disrupted CD70 gene. In someembodiments, the T cells are human T cells. In some embodiments, theengineered immune cells (e.g., T cells) comprise a disrupted TRAC gene,a disrupted B2M gene, a disrupted CD70 gene, and a nucleic acid encodinga CAR. In some embodiments, the disrupted TRAC gene comprises thenucleic acid encoding the CAR. In some embodiments the engineered immunecell (e.g., T cell) further comprises a disrupted PD-1 gene. In someembodiments the nucleic acid encoding a CAR target a tumor antigen(e.g., BCMA, CD19, CD33 or CD70).

In some aspects the engineered immune cell (e.g., T cell) providedexhibits improved T cell function including the prevention of prematureexhaustion, enhanced CAR T cell expansion, and increased efficiency ofcancer cell killing. In some aspects the engineered immune cell (e.g., Tcell) provided exhibit continued, steady cell growth, relative tounedited T cells or relative to edited T cells that express CD70, aswell as showing increased cytotoxicity and cytokine (e.g., IL-2 and/orIFN-gamma) secretion.

In some aspects, the disclosure provides an engineered T cell comprisinga disrupted CD70 gene and a nucleic acid encoding a CAR that does notbind CD70. In some aspects, the engineered T cell comprises a disruptedT cell receptor alpha constant region (TRAC) gene. In some aspects, thedisrupted TRAC gene comprises the nucleic acid encoding the CAR thatdoes not bind CD70. In some aspects, the engineered T cell comprises adisrupted beta-2-microglobulin (β2M) gene.

In some aspects, the disclosure provides an engineered T cellcomprising: (i) a disrupted TRAC gene; (ii) a disrupted B2M gene; (iii)a disrupted CD70 gene; and (iv) a nucleic acid encoding a CAR that doesnot bind CD70.

In some aspects, the disclosure provides a population of cellscomprising engineered T cells, wherein the engineered T cells comprise adisrupted CD70 gene and a nucleic acid encoding a CAR that does not bindCD70.

In some aspects, the engineered T cell in the population of cellscomprises a disrupted T cell receptor alpha constant region (TRAC) gene.In some aspects, the disrupted TRAC gene comprises the nucleic acidencoding the CAR that does not bind CD70. In some aspects, theengineered T cell in the population of cells comprises a disruptedbeta-2-microglobulin (β2M) gene.

In some aspects, the disclosure provides a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:(i) a disrupted TRAC gene; (ii) a disrupted B2M gene; (iii) a disruptedCD70 gene; and (iv) a nucleic acid encoding a CAR that does not bindCD70.

In any of the foregoing or related aspects, the CAR comprises anectodomain that binds i-B cell maturation antigen (BCMA). In someaspects, the ectodomain comprises an anti-BCMA antibody. In someaspects, the ectodomain comprises an anti-BCMA single-chain variablefragment (scFv). In some aspects, the anti-BCMA scFv comprises variableheavy (VH) chain complementarity determining regions (CDRs) and the samevariable light (VL) chain CDRs as a reference antibody, wherein thereference antibody comprises a VH set forth as SEQ ID NO: 60 and a VLset forth as SEQ ID NO: 61. In some aspects, the anti-BCMA scFvcomprises VH and VL chains comprising the amino acid sequences set forthin SEQ ID NOs: 60 and 61, respectively. In some aspects, the anti-BCMAscFv comprises the amino acid sequence of SEQ ID NO: 59. In someaspects, the anti-BCMA scFv is encoded by a nucleotide sequence havingat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:57.

In any of the foregoing or related aspects, the anti-BCMA scFv comprisesthe amino acid sequence of SEQ ID NO: 59. In some embodiments, theanti-BCMA scFv comprises a VH comprising the amino acid sequence of SEQID NO: 60. In some embodiments, the anti-BCMA scFv comprises a VLcomprising the amino acid sequence of SEQ ID NO: 61. In someembodiments, the anti-BCMA scFv comprises a VH comprising CDR amino acidsequences of (i) SEQ ID NO: 80, SEQ ID NO: 82, and/or SEQ ID NO: 84 or(ii) SEQ ID NO: 81, SEQ ID NO: 83, or SEQ ID NO: 85; and/or theanti-BCMA scFv comprises a VL sequence comprising CDR amino acidsequences of (i) SEQ ID NO: 74, SEQ ID NO: 76, and/or SEQ ID NO: 78.

In any of the foregoing or related aspects, the CAR comprises anectodomain that binds CD33. In some the ectodomain comprises ananti-CD33 antibody. In some aspects, the ectodomain comprises ananti-CD33 scFv. In some aspects, the anti-CD33 scFv comprises the sameVH CDRs and the same VL chain CDRs as a reference antibody, wherein thereference antibody comprises a VH set forth as SEQ ID NO: 140 and a VLset forth as SEQ ID NO: 141. In some aspects, the anti-CD33 scFvcomprises VH and VL chains comprising the amino acid sequences set forthin SEQ ID NOs: 140 and 141, respectively. In some aspects, the anti-CD33scFv comprises the amino acid sequence of SEQ ID NO: 137.

In any of the foregoing or related aspects, the CAR comprises anectodomain that binds CD19. In some aspects, wherein the ectodomaincomprises an anti-CD19 antibody. In some aspects, the ectodomaincomprises an anti-CD19 scFv. In some aspects, the anti-CD19 scFvcomprises the same VH CDRs and the same VL chain CDRs as a referenceantibody, wherein the reference antibody comprises a VH set forth as SEQID NO: 152 and a VL set forth as SEQ ID NO: 153. In some aspects, theanti-CD19 scFv comprises VH and VL chains comprising the amino acidsequences set forth in SEQ ID NOs: 152 and 153, respectively. In someaspects, the anti-CD19 scFv comprises the amino acid sequence of SEQ IDNO: 151.

In some aspects, the disclosure provides an engineered T cellcomprising: (i) a disrupted TRAC gene; (ii) a disrupted B2M gene; (iii)a disrupted CD70 gene; and (iv) a nucleic acid encoding a CAR that bindsCD70. In some aspects, the disrupted TRAC gene comprises the nucleicacid encoding the CAR.

In some aspects, the disclosure provides a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:(i) a disrupted TRAC gene; (ii) a disrupted B2M gene; (iii) a disruptedCD70 gene; and (iv) a nucleic acid encoding a CAR that binds CD70.

In some aspects, the disclosure provides a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene;

(ii) a disrupted β2M gene;

(iii) a disrupted CD70 gene

(iv) a nucleic acid encoding a CAR comprising (a) an ectodomain thatcomprises an anti-CD70 scFv, (b) a CD8 transmembrane domain, and (c) anendodomain that comprises a 41BB co-stimulatory domain and a CD3zsignaling domain.

In any of the foregoing or related aspects, the CAR that binds CD70comprises an ectodomain comprising an anti-CD70 antibody. In someaspects, CAR comprises an ectodomain comprising an anti-CD70 scFv. Insome aspects, the anti-CD70 scFv comprises the same VH CDRs and the sameVL CDRs as a reference antibody, wherein the reference antibodycomprises a VH set forth as SEQ ID NO: 51 and a VL set forth as SEQ IDNO: 52. In some aspects, the anti-CD70 scFv comprises VH and VL chainscomprising the amino acid sequences set forth in SEQ ID NOs: 51 and 52,respectively. In some aspects, the anti-CD70 scFv comprises the aminoacid sequence of SEQ ID NO: 48 or 50. In some aspects, the anti-CD70scFv comprises the amino acid sequence of SEQ ID NO: 50.

In any of the foregoing or related aspects, the anti-CD70 scFv comprisesa VH comprising the amino acid sequence of SEQ ID NO: 51. In someembodiments, the anti-CD70 scFv comprises a VL comprising the amino acidsequence of SEQ ID NO: 52. In some embodiments, the anti-CD70 scFvcomprises a VH comprising CDR amino acid sequences of (i) SEQ ID NO: 68,SEQ ID NO: 70, and/or SEQ ID NO: 72 or (ii) SEQ ID NO: 69, SEQ ID NO:71, and/or SEQ ID NO: 73; and/or the anti-CD70 scFv comprises a VLsequence comprising CDR amino acid sequences of (i) SEQ ID NO: 62, SEQID NO: 64, and/or SEQ ID NO: 66 or (ii) SEQ ID NO: SEQ ID NO: 63, SEQ IDNO: 65, and/or SEQ ID NO: 67.

In any of the foregoing or related aspects, the CAR comprises a CD28 or41BB co-stimulatory domain. In any of the foregoing or related aspects,the CAR comprises a CD3ζ signaling domain. In any of the foregoing orrelated aspects, the CAR comprises a CD8 transmembrane domain.

In any of the foregoing or related aspects, there is a deletion in theTRAC gene relative to unmodified T cells. In some aspects, the deletionis 15-30 base pairs. In some aspects, the deletion is 20 base pairs. Insome aspects, the deletion comprises SEQ ID NO: 86. In some aspects, thedeletion is of SEQ ID NO: 86.

In some aspects, the disclosure provides an engineered T cell comprisinga disrupted CD70 gene and a nucleic acid encoding a CAR that binds CD70,wherein the CAR comprises the amino acid sequence set forth in SEQ IDNO: 46. In some aspects, the disclosure provides an engineered T cellcomprising a disrupted CD70 gene, and a nucleic acid encoding a CAR thatbinds CD70, wherein the nucleic acid sequence is at least 90% identicalto SEQ ID NO: 45. In some aspects, the disclosure provides an engineeredT cell comprising a disrupted CD70 gene, and a nucleic acid encoding aCAR that binds CD70, wherein the nucleic acid sequence is SEQ ID NO: 45.

In some embodiments, the CD70 gene is disrupted by CRISPR/Cas9 geneediting. In some embodiments, the TRAC gene is disrupted by CRISPR/Cas9gene editing. In some embodiments, the B2M gene is disrupted byCRISPR/Cas9 gene editing. In some embodiments, the PD-1 gene isdisrupted by CRISPR/Cas9 gene editing.

In some aspects, the disclosure provides an engineered T cellcomprising:

-   -   (i) a disrupted TRAC gene, wherein the disrupted TRAC gene        comprises a nucleic acid encoding a CAR comprising the amino        acid sequence set forth in SEQ ID NO: 46;    -   (ii) a disrupted B2M gene; and    -   (iii) a disrupted CD70 gene. In some embodiments, the nucleic        acid encoding the CAR comprises a sequence at least 80%, at        least 90%, at least 95%, at least 96%, at least 97%, at least        98% or at least 99% identical to SEQ ID NO: 45.

In other aspects, the disclosure provides an engineered T cellcomprising:

-   -   (i) a disrupted TRAC gene, wherein the disrupted TRAC gene        comprises a nucleic acid encoding a CAR, wherein the nucleic        acid sequence is at least 90% identical to SEQ ID NO: 45;    -   (ii) a disrupted B2M gene; and    -   (iii) a disrupted CD70 gene. In some embodiments, the disrupted        TRAC gene comprises a donor sequence comprising the nucleotide        sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 44.

In some aspects, the disclosure provides an engineered T cellcomprising:

-   -   (i) a disrupted TRAC gene comprising a nucleic acid sequence at        least 90% identical to SEQ ID NO: 44;    -   (ii) a disrupted B2M gene; and    -   (iii) a disrupted CD70 gene.

In some aspects, the disclosure provides an engineered T cellcomprising:

-   -   (i) a disrupted TRAC gene comprising the nucleic acid sequence        of SEQ ID NO: 44;    -   (ii) a disrupted B2M gene; and    -   (iii) a disrupted CD70 gene.

In any of the foregoing or related aspects, the engineered T cellcomprises a disrupted PD-1 gene.

In any of the foregoing or related aspects, the engineered T cellmaintains cytotoxicity following 5 rechallenges with a target cell,wherein the target cell expresses an antigen specific for the CAR. Insome aspects, the engineered T cell maintains cytotoxicity following 10rechallenges with the target cell. In some aspects, the target cell is acancer cell. In some aspects, the target cell is a cancer cell of ahematological cancer or solid tumor.

In any of the foregoing or related aspects, the engineered T cell orpopulation of cells comprises a CAR comprising the amino acid sequenceof SEQ ID NO: 57. In some aspects, the CAR is encoded by a nucleic acidsequence having at least 90% identity to SEQ ID NO: 56.

In any of the foregoing or related aspects, the engineered T cell orpopulation of cells comprises a CAR comprising the amino acid sequenceof SEQ ID NO: 139. In some aspects, the CAR is encoded by a nucleic acidsequence having at least 90% identity to SEQ ID NO: 136.

In any of the foregoing or related aspects, the engineered T cell orpopulation of cells comprises a CAR comprising the amino acid sequenceof SEQ ID NO: 149. In some aspects, the CAR is encoded by a nucleic acidsequence having at least 90% identity to SEQ ID NO: 148.

In any of the foregoing or related aspects, the engineered T cell orpopulation of cells comprises a CAR comprising the amino acid sequenceof SEQ ID NO: 46. In some aspects, the CAR is encoded by a nucleic acidsequence having at least 90% identity to SEQ ID NO: 45.

Other aspects of the present disclosure provide a population of cellscomprising any of the engineered immune cells (e.g., T cells) describedherein. In some embodiments, a population of cells comprise T cells thatcomprise a TRAC gene disrupted by insertion of a nucleic acid encoding aCAR, a disrupted β2M gene, and a disrupted CD70 gene. In someembodiments, a population of cells comprise T cells that comprise adisrupted TRAC gene, a disrupted B2M gene, a disrupted CD70 gene, and anucleic acid encoding a CAR. In some embodiments, a population of cellscomprise T cells that comprise a disrupted TRAC gene, wherein thedisrupted TRAC gene comprises a nucleic acid encoding a CAR, a disruptedB2M gene, and a disrupted CD70 gene.

In some aspects, the disclosure provides a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:

-   -   (i) a disrupted TRAC gene, wherein the disrupted TRAC gene        comprises a nucleic acid encoding a CAR comprising (a) an        ectodomain that comprises an anti-CD70 antigen-binding        fragment, (b) a CD8 transmembrane domain, and (c) an endodomain        that comprises a 41BB co-stimulatory domain and a CD3z signaling        domain;    -   (ii) a disrupted beta-2-microglobulin (B2M) gene; and    -   (iii) a disrupted CD70 gene.

In some aspects, the disclosure provides a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises anucleic acid encoding a CAR comprising the amino acid sequence set forthin SEQ ID NO: 46;

(ii) a disrupted β2M gene; and

(iii) a disrupted CD70 gene.

In other aspects, the disclosure provides a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises anucleic acid encoding a CAR, wherein the nucleic acid sequence is atleast 90% identical to SEQ ID NO: 45;

(ii) a disrupted β2M gene; and

(iii) a disrupted CD70 gene. In some aspects, the disrupted TRAC genecomprises the nucleic acid sequence set forth in SEQ ID NO: 45.

In some aspects, the disclosure provides a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene comprising a nucleic acid sequence at least90% identical to SEQ ID NO: 44;

(ii) a disrupted β2M gene; and

(iii) a disrupted CD70 gene. In some aspects, the disrupted TRAC genecomprises the nucleic acid sequence set forth in SEQ ID NO: 44.

In some embodiments, the CAR comprises a CD3z signaling domain. In someembodiments, the CAR comprises a CD8 transmembrane domain. In someembodiments, the CAR comprises a CD28 or 41BB co-stimulatory domain.

In any of the foregoing or related aspects of the population of cells,the disrupted β2M gene comprises at least one nucleotide sequenceselected from any one of SEQ ID NOS: 9-14. In any of the foregoing orrelated aspects of the population of cells, the disrupted CD70 genecomprises at least one nucleotide sequence selected from any one of SEQID NOS: 129-134.

In some embodiments, the TRAC gene comprises the nucleotide sequence ofSEQ ID NO: 45 and/or the nucleic acid encoding the anti-CD70 CARcomprises the nucleotide sequence of SEQ ID NO: 45. In some embodiments,the TRAC gene comprises the nucleotide sequence of SEQ ID NO: 45. Insome embodiments, the TRAC gene comprises the nucleotide sequence of SEQID NO: 44. In some embodiments, the TRAC gene comprises the nucleotidesequence of SEQ ID NO: 56 and/or the nucleic acid encoding the anti-BCMACAR comprises the nucleotide sequence of SEQ ID NO: 56. In someembodiments, the TRAC gene comprises the nucleotide sequence of SEQ IDNO: 56. In some embodiments, the TRAC gene comprises the nucleotidesequence of SEQ ID NO: 55.

In some embodiments, the TRAC gene comprises the nucleotide sequence ofSEQ ID NO: 156 and/or the nucleic acid encoding the anti-CD19 CARcomprises the nucleotide sequence of SEQ ID NO: 148. In someembodiments, the TRAC gene comprises the nucleotide sequence of SEQ IDNO: 148. In some embodiments, the TRAC gene comprises the nucleotidesequence of SEQ ID NO: 156. In some embodiments, the TRAC gene comprisesthe nucleotide sequence of SEQ ID NO: 135 and/or the nucleic acidencoding the anti-CD33 CAR comprises the nucleotide sequence of SEQ IDNO: 136. In some embodiments, the TRAC gene comprises the nucleotidesequence of SEQ ID NO: 136. In some embodiments, the TRAC gene comprisesthe nucleotide sequence of SEQ ID NO: 135.

In any of the foregoing aspects, the engineered T cells:(a) exhibitincreased cellular proliferative capacity;

(b) exhibit increased cell lysis;

(c) exhibit reduced cellular exhaustion;

(d) maintain cytokine-dependent proliferation;

(e) exhibit increased cytokine secretion; or

(f) any combination of (a)-(e),

relative to control T cells, wherein control T cells express endogenousCD70 protein.

In some embodiments, at least 50%, optionally 50%-65%, of the engineeredT cells do not express a detectable level of TCR surface protein, do notexpress a detectable level of β2M surface protein, do not express adetectable level of CD70 surface protein, and/or express a detectablelevel of the CAR.

In some embodiments, at least 90%, optionally 90%-100%, of theengineered T cells do not express a detectable level of TCR surfaceprotein. In some embodiments, greater than 99.5% of the engineered Tcells do not express a detectable level of TCR surface protein.

In some embodiments, at least 60%, optionally 60%-75%, of the engineeredimmune cells (e.g., T cells) do not express a detectable level of β2Msurface protein.

In some embodiments, at least 80%, optionally 80%-100%, of theengineered immune cells (e.g., T cells) do not express a detectablelevel of CD70 surface protein.

In some embodiments, at least 80%, optionally 80%-95%, of the engineeredimmune cells (e.g., T cells) express a detectable level of the CAR(e.g., an anti-CD70 CAR or an anti-BCMA CAR).

In some embodiments, the engineered immune cells (e.g., T cells) furthercomprise a disrupted PD-1 gene.

In some embodiments, at least 50%, optionally 50%-70%, of the engineeredT cells do not express a detectable level of TCR surface protein, do notexpress a detectable level of β2M surface protein, do not express adetectable level of PD-1 surface protein, do not express a detectablelevel of CD70 surface protein, and/or express a detectable level of theCAR.

-   -   In some aspects, the disclosure provides a method for producing        an engineered T cell, the method comprising:    -   (a) delivering to a T cell    -   an RNA-guided nuclease,    -   a gRNA targeting a CD70 gene, and    -   a vector comprising a donor template that comprises a nucleic        acid encoding a CAR; and    -   (b) producing an engineered T cell comprising a disrupted CD70        gene and expressing the CAR.

In some aspects, the method further comprises delivering to the T cell agRNA targeting a TRAC gene; wherein the engineered T cell furthercomprises a disrupted TRAC gene. In some aspects, the nucleic acidencoding the CAR is flanked by left and right homology arms to the TRACgene; and wherein the engineered T cell comprises the nucleic acidencoding the CAR in the TRAC gene. In some aspects, the method furthercomprises delivering to the T cell a gRNA targeting a β2M gene; whereinthe engineered T cell of further comprises a disrupted β2M gene.

Also provided herein are methods for producing an engineered T cell, themethod comprising (a) delivering to a T cell an RNA-guided nuclease, agRNA targeting a TRAC gene, a gRNA targeting a β2M gene, a gRNAtargeting a CD70 gene, and a vector comprising a donor template thatcomprises a nucleic acid encoding a CAR, optionally wherein the nucleicacid encoding the CAR is flanked by left and right homology arms to theTRAC gene locus, and (b) producing an engineered T cell.

In some embodiments, the RNA-guided nuclease is a Cas9 nuclease,optionally a Streptococcus pyogenes Cas9 nuclease. Other RNA-guidednucleases may be used and are described below.

In some embodiments, wherein the gRNA targeting the TRAC gene comprisesthe nucleotide sequence of SEQ ID NO: 98 or targets the nucleotidesequence of SEQ ID NO: 118, and optionally wherein the gRNA targetingthe TRAC gene comprises the nucleotide sequence of SEQ ID NO: 30. Insome embodiments, the gRNA targeting the β2M gene comprises thenucleotide sequence of SEQ ID NO: 99 or targets the nucleotide sequenceof SEQ ID NO: 119, and optionally wherein the gRNA targeting the β2Mgene comprises the nucleotide sequence of SEQ ID NO: 31. In someembodiments, the gRNA targeting the CD70 gene comprises the nucleotidesequence of SEQ ID NOS: 94 or 95 or targets the nucleotide sequence ofSEQ ID NO: 114 or 115, and optionally wherein the gRNA targeting theCD70 gene comprises the nucleotide sequence of SEQ ID NOS: 26 or 27.

In any of the foregoing aspects, the RNA-guided nuclease and gRNA arecomplexed in a ribonucleorotein particle (RNP).

In some embodiments, the methods further comprise delivering to the Tcell a gRNA targeting a PD-1 gene. In some aspects, the engineeredimmune cells are allogeneic T cells comprising a TRAC gene disrupted byinsertion of a nucleic acid encoding a CAR, a disrupted β2M gene, and adisrupted PD-1 gene. In some embodiments the engineered immune cell(e.g., T cell) further comprises a disrupted CD70 gene.

In some embodiments, the gRNA targeting the PD-1 gene comprises thenucleotide sequence of SEQ ID NO: 100 or targets the nucleotide sequenceof SEQ ID NO: 120, and optionally wherein the gRNA targeting the PD-1gene comprises the nucleotide sequence of SEQ ID NO: 32.

In some aspects, the disclosure provides a method for producing anengineered T cell for immunotherapy against a target cell, comprising:

(a) disrupting a CD70 gene in a T cell, and

(b) expressing a CAR that binds to an antigen expressed on the targetcell, wherein the antigen is not CD70. In some aspects, the target cellis a cancer cell. In some aspects, the method is ex vivo. In someaspects, the method further comprises comprising disrupting a TRAC genein the T cell. In some aspects, the method further comprises disruptinga β2M gene in the T cell. In some aspects, the CAR is encoded by anucleic acid in the disrupted TRAC gene. In some aspects, the CAR is anyone of the CARs described herein.

In some aspects, the disclosure provides a population of engineered Tcells produced by any one of the methods described herein.

In some aspects, the disclosure provides a method of increasingproliferation of T cells, comprising disrupting the CD70 gene in the Tcells. In some aspects, the disclosure provides a method of reducingexhaustion of T cells, comprising disrupting the CD70 gene in the Tcells. In any of the foregoing aspects, the CD70 gene is disrupted byCRISPR/Cas gene editing. In some aspects, the method further comprisesdisrupting the TRAC gene, the β2M gene, or both the TRAC and β2M genesin the T cells. In some aspects, the TRAC gene, β2M gene or both TRACand β2M gene is disrupted by CRISPR/Cas gene editing.

In some embodiments, the vector comprises a nucleic acid encoding a CARthat comprises the amino acid sequence of SEQ ID NO: 46. In someembodiments, the vector comprises a nucleic acid encoding a CAR thatcomprises the amino acid sequence of SEQ ID NO: 57. In some embodiments,the vector comprises a nucleic acid encoding a CAR that comprises theamino acid sequence of SEQ ID NO: 149. In some embodiments, the vectorcomprises a nucleic acid encoding a CAR that comprises the amino acidsequence of SEQ ID NO: 139.

In some aspects, the disclosure provides methods for administering thepopulation of cells or an engineered T cells described herein to asubject. In some aspects, the engineered T cells are engineered human Tcells. In some aspects, the subject has cancer. In some aspects, thecancer expresses CD70, BMCA, CD19, CD33 or combinations thereof. In someaspects, the population of cells is administered to the subject in anamount effective to treat the cancer. In some aspects, the cancer is asolid tumor malignancy or a hematological malignancy. In some aspects,the solid tumor malignancy is selected from the group consisting of:ovarian tumor, pancreatic tumor, kidney tumor, lung tumor, andintestinal tumor. In some aspects, the population of cells isadministered to the subject in an amount effective to reduce the volumeof a tumor in the subject.

In some aspects, the disclosure provides a method for treating cancer ina subject, comprising administering the population of cells or anengineered T cells described herein to a subject.

In some aspects, the disclosure provides a method for treating cancer ina subject, comprising administering to the patient a population of cellscomprising engineered T cells, wherein the engineered T cells comprise adisrupted CD70 gene and a nucleic acid encoding a CAR, thereby treatingcancer in the subject. In some embodiments, the CAR binds CD70. In someembodiments, the CAR does not bind CD70.

In other aspects, the disclosure provides a method for treating cancerin a subject, comprising administering to the patient a population ofcells comprising engineered T cells, wherein the engineered T cellscomprise:

(i) a disrupted TRAC gene;;

(ii) a disrupted B2M gene;

(iii) a disrupted CD70 gene; and

(iv) a nucleic acid encoding a CAR;

thereby treating the cancer in the subject.

In yet other aspects, the disclosure provides a method for treatingcancer in a subject, comprising administering to the patient apopulation of cells comprising engineered T cells, wherein theengineered T cells comprise:

(i) a disrupted TRAC gene;

(ii) a disrupted B2M gene;

(iii) a disrupted CD70 gene; and

(iv) a nucleic acid encoding a CAR comprising (a) an ectodomain thatcomprises an anti-CD70 antigen-binding fragment, (b) a CD8 transmembranedomain, and (c) an endodomain that comprises a 41BB co-stimulatorydomain and a CD3z signaling domain,

thereby treating the cancer in the subject. In some embodiments, the CARcomprises the amino acid sequence of SEQ ID NO: 46. In some embodiments,the nucleic acid encoding the CAR comprises the nucleotide sequence ofSEQ ID NO: 45. In some embodiments, the disrupted TRAC gene comprisesthe nucleotide sequence of SEQ ID NO: 45 or SEQ ID NO: 44.

In some aspects, the disclosure provides a method of treating cancer ina subject, comprising administering to the subject a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises anucleic acid encoding a CAR comprising the amino acid sequence set forthin SEQ ID NO: 46;

(ii) a disrupted β2M gene; and

(iii) a disrupted CD70 gene,

thereby treating the cancer in the subject.

In some aspects, the disclosure provides a method of treating cancer ina subject, comprising administering to the subject a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises anucleic acid encoding a CAR, wherein the nucleic acid sequence is atleast 90% identical to SEQ ID NO: 45;

(ii) a disrupted β2M gene; and

(iii) a disrupted CD70 gene,

thereby treating the cancer in the subject. In some aspects, thedisrupted TRAC gene comprises the nucleic acid sequence set forth in SEQID NO: 45.

In some aspects, the disclosure provides a method of treating cancer ina subject, comprising administering to the subject a population of cellscomprising engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene comprising a nucleic acid sequence at least90% identical to SEQ ID NO: 44;

(ii) a disrupted β2M gene; and

(iii) a disrupted CD70 gene,

thereby treating the cancer in the subject. In some aspects, thedisrupted TRAC gene comprises the nucleic acid sequence set forth in SEQID NO: 44.

In any of the foregoing or related aspects, the engineered T cells areengineered human T cells. In some embodiments, the engineered T cellsare engineered allogeneic T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes a graph showing highly efficient multiple gene editingin TRAC−/β2M−/PD-1−/CD70− (quadruple knockout) T cells.

FIG. 2 includes a graph showing similar expansion among multigene-editedcells.

FIG. 3 includes graphs showing efficient multiple gene editing inTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ (i.e., 3× KO (CD70), CD70 CAR⁺) T cells.

FIG. 4 includes a graph showing that normal proportions of CD4+ and CD8+T cells are maintained among the TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cellpopulation.

FIG. 5 includes a graph showing efficient multiple gene editing inTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells.

FIG. 6 includes a graph showing that normal proportions of CD4+ and CD8+T cells are maintained among the TRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70CAR⁺(i.e., 4× KO, CD70 CAR⁺) T cell population.

FIGS. 7A-7C include graphs showing data relating to the characterizationof anti-BCMA CAR+ T cells with multi-gene edits. Double knockoutTRAC⁻/β2M⁻/anti-BCMA CAR⁺ T cells and quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-BCMA CAR⁺ T cells were stained for TRAC andβ2M (FIG. 7A), PD-1 and CD70 (FIG. 7B), and BCMA CAR (FIG. 7C)expression. The anti-BCMA CAR was expressed at approximately 80% in boththe double and quadruple knockout CAR T cells.

FIG. 8 includes flow cytometry plots showing prevention of loss of CD4+cells in 3× KO (TRAC−/β2M−/CD70−) anti-CD33 CAR T cells compared 2× KO(TRAC−/β2M−) anti-CD33 CAR T cells over three weeks.

FIG. 9 includes a graph showing CD70 KO enhanced cell proliferation inanti-CD33 CAR T cells over two weeks. The total number of viable cellswas quantified in 3× KO (TRAC−/β2M−/CD70−) and 2× KO (TRAC−/β2M−)anti-CD33 CAR T cells.

FIG. 10 includes a graph showing CD70 KO enhanced cell proliferation inanti-CD19 CAR T cells over two weeks. The total number of viable cellswas quantified in 3× KO (TRAC−/β2M−/CD70−) and 2× KO (TRAC−/β2M−)anti-CD33 CAR T cells.

FIG. 11 includes graphs showing CD70 KO enhanced cell proliferation inanti-BCMA CAR T cells and rescued the detrimental effect of PD1 KO onBCMA CAR cell proliferation. The total number of viable cells wasquantified in 4× KO (TRAC−/β2M−/CD70−/PD1−), 3× KO (CD70)(TRAC−/β2M−/CD70−), 3× KO (PD1) (TRAC−/β2M−/PD1−) and 2× KO (TRAC−/β2M−)anti-CD33 CAR T cells.

FIG. 12 includes graphs showing CD70 KO enhanced cell proliferation inanti-BCMA CAR T cells and rescued the detrimental effect of PD1 KO onBCMA CAR cell proliferation. The total number of viable cells wasquantified in 4× KO (TRAC−/β2M−/CD70−/PD1−), 3× KO (CD70)(TRAC−/β2M−CD70−), 3× KO (PD1) (TRAC−/β2M−/PD1−) and 2× KO (TRAC−/β2M−)anti-CD33 CAR T cells. The anti-BCMA CAR T cells were derived from adifferent donor T cells as the CAR T cells shown in FIG. 11.

FIG. 13 includes a graph showing a comparison of apoptotic cell deathdue to antigen exposure in 2× KO (TRAC−/β2M−) anti-BCMA CAR+ T cells and3× KO (TRAC−/β2M−/CD70−) anti-BCMA CAR+ T cells. CAR+ T cells wereexposed to plate-bound BCMA antigen for 24 hours with a re-challengeevery 24 hours and apoptosis was assessed following each antigenchallenge by flow cytometry. Induction of apoptosis due to antigenchallenge was lower in anti-BCMA CAR+ T cells with a CD70 KO compared tothose without.

FIG. 14 includes a graph showing a comparison of CAR T cell expansionfollowing antigen exposure in 2× KO (TRAC−/β2M−) anti-BCMA CAR+ T cellsand 3× KO (TRAC−/β2M−/CD70−) anti-BCMA CAR+ T cells. CAR+ T cells wereexposed to plate-bound BCMA antigen for 24 hours with a re-challengeevery 24 hours and cell expansion was assessed following each antigenchallenge and normalized to the population at time 0 h. Populationexpansion following antigen challenge was higher in anti-BCMA CAR+ Tcells with a CD70 KO compared to those without.

FIG. 15 includes a graph showing robust cell expansion inTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells. The total number of viablecells was quantified in 3× KO (TRAC−/β2M−/CD70−) and 2× KO (TRAC−/β2M−)anti-CD70 CAR T cells. 3× KO cells were generated with either CD70 sgRNAT7 or T8.

FIG. 16 includes a graph showing robust cell expansion ofTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CA⁺ T cells. The total number of viablecells was quantified in 4× KO (TRAC−/β2M−/PD1−/CD70−), 3× KO(TRAC−/β2M−/PD1−) and 2× KO (TRAC−/β2M−) anti-CD70 CAR T cells.

FIG. 17 includes graphs showing robust cell killing of both Nalm6 (toppanel) cells and Raji (bottom panel) cells by anti-CD19 CAR T cells(TRAC⁻/β2M⁻/CD70⁻/anti-CD19 CAR⁺ or TRAC⁻/β2M⁻/anti-CD19 CAR⁺ T cells).

FIG. 18 includes a graph showing robust cell killing of MV411 cells byanti-CD33 CAR T cells (TRAC⁻/β2M⁻/CD70⁻/anti-CD33 CAR⁺ orTRAC⁻/β2M⁻/anti-CD33 CAR⁺ T cells).

FIG. 19 includes a graph showing robust cell killing of A498 cells by 3×KO (TRAC⁻/β2M⁻/CD70⁻) anti-CD70 CAR⁺ T cells compared to 2× KO(TRAC⁻/β2M⁻) anti-CD70 CAR⁺ T cells.

FIG. 20 includes a graph showing cell expansion of 3× KO(TRAC−/β2M−/CD70−) or 2× KO (TRAC−/β2M−) anti-CD33 CAR T cells afterchallenge with MV411 target cells.

FIG. 21 includes a graph showing cell expansion of 3× KO(TRAC−/β2M−/CD70−) or 2× KO (TRAC−/β2M−) anti-CD70 CAR T cells afterchallenge with Nalm6 target cells.

FIG. 22A includes a graph showing A498 cell killing by anti-CD70 CAR Tcells after serial rechallenge. 4× KO (TRAC⁻/β2M⁻/CD70⁻/PD1⁻), 3× KO(CD70) (TRAC⁻/β2M⁻/CD70⁻), 3× KO (PD1) (TRAC⁻/β2M⁻/PD1⁻) and 2× KO(TRAC⁻/β2M⁻) anti-CD70 CAR+ T cells were utilized. 3× KO (CD70), CD70CAR⁺ T cells, and 4× KO, CD70 CAR+ T cells were the most effective. FIG.22B includes a graph showing ACHN cell killing by anti-CD70 CAR T cellsafter serial rechallenge. The same cells as FIG. 22A were utilized. 3×KO (CD70), CD70 CAR⁺ T cells and 4× KO, CD70 CAR⁺ T cells were the mosteffective. FIG. 23A includes a graph showing ACHN cell killing byanti-CD70 CAR T cells at various effector:target ratios. 4× KO(TRAC⁻/β2M⁻/CD70⁻/PD1⁻), 3× KO (CD70) (TRAC⁻/β2M⁻/CD70⁻), 3× KO (PD1)(TRAC⁻/β2M⁻/PD1⁻) and 2× KO (TRAC⁻/β2M⁻) anti-CD70 CAR+ T cells wereutilized. 3× KO (CD70), CD70 CAR⁺ T cells and 4× KO, CD70 CAR⁺ T cellswere superior killers following multiple serial rechallenges. FIG. 23Bincludes a graph showing LAG3 (left) and PD1 (right) expression in thecells from FIG. 23A following eight rechallenges.

FIGS. 24A-24C include graphs showing that knockout of PD-1 and CD70enhances cell killing activity of anti-BCMA CAR+ T cells as measuredthrough serial rechallenges with a multiple myeloma cell line (MM.1S).Double knockout (2× KO (TRAC⁻/β2M⁻) anti-BCMA CAR⁺ T cells (circles)began to lose their potency towards MM.1S cells after approximately 4rechallenges, while quadruple knockout (4× KO (TRAC⁻/β2M⁻/CD70⁻/PD1⁻)anti-BCMA CAR⁺ T cells (squares) were capable of killing 100% of theMM.1S cells after 10 rechallenges (FIG. 24A). Consistent with this, thequadruple knockout anti-BCMA CAR⁺ T cells continued to secrete IFN-g inresponse to target cells after 10 rechallenges, while the doubleknockout anti-BCMA CAR⁺ T cells showed reduced IFN-g secretion after thethird rechallenge (FIG. 24B). The quadruple knockout anti-BCMA CAR⁺ Tcells also showed higher proliferation in response to exposure to targetcells than the double knockout anti-BCMA CAR⁺ T cells (FIG. 24C).

FIGS. 25A-25C include graphs showing highest cell kill activity in A498PD-L1 kidney cancer cells (which overexpress PD-L1) using quadrupleknockout (4× KO) TRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells andtriple knockout (3× KO (CD70)) TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells,relative to double knockout (2× KO) TRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cellsand triple knockout (3× KO (PD1) TRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ Tcells. A CAR T cell:A498-PD-L1 cell ratio of 2:1 was used in FIG. 25A, aCAR T cell:A498-PD-L1 cell ratio of 1:1 was used in FIG. 25B, and a CARTcell:A498-PD-L1 cell ratio of 0.5:1 was used in FIG. 25C.

FIG. 26A and FIG. 26B include graphs showing that quadruple knockout (4×KO) TRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells secrete the highestlevels of cytokines IFN-gamma (FIG. 26A) and IL-2 (FIG. 26B), relativeto triple knockout (3× KO (CD70) TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ Tcells, double knockout (2× KO) TRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cells andtriple knockout (3× KO (PD1) TRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ T cells. ACART cell:A498-PD-L1 cell ratio of 1:1 was used.

FIG. 27A includes a graph showing results from an experiment designed toassess tumor volume reduction in a subcutaneous A498 renal cellcarcinoma model exposed to: 2× KO (TRAC⁻/β2M⁻), CD70 CAR⁺ T cells; 3× KO(PD-1) (TRAC⁻/β2M⁻/PD1⁻), CD70 CAR⁺ T cells; 3× KO (CD70)(TRAC⁻/β2M⁻/CD70⁻), CD70 CAR⁺ T cells; or 4× KO (PD-1, CD70)(TRAC⁻/β2M⁻/PD1⁻/CD70⁻), CD70 CAR⁺ T cells. FIG. 27B includes a graphshowing results from an experiment designed to assess prevention oftumor growth in a subcutaneous A498 renal cell carcinoma rechallengemodel. Mice from FIG. 27A were rechallenged with A498 tumor cells on day25 and tumor volume was assessed over time. FIG. 27C includes a graphshowing results from an experiment designed to assess tumor volumereduction in a subcutaneous A498 renal cell carcinoma model (large tumorof ˜150 mm3 at time of CAR-T injection) exposed to: 3× KO (CD70)(TRAC⁻/β2M⁻/CD70⁻), CD70 CAR⁺ T cells; 4× KO (PD-1, CD70)(TRAC⁻/β2M⁻/PD1⁻/CD70⁻), CD70 CAR⁺ T cells; 2× KO (TRAC⁻/β2M⁻), CD70CAR⁺ T cells; or 3× KO (PD-1) (TRAC⁻/β2M⁻/PD1⁻), CD70 CAR⁺ T cells.

FIG. 28A includes a graph showing tumor volume reduction in asubcutaneous MM.1S model exposed to: 2× KO (TRAC⁻/β2M⁻), BCMA CAR⁺ Tcells; 3× KO (PD-1) (TRAC⁻/β2M⁻/PD1⁻), BCMA CAR⁺ T cells; 3× KO (CD70)(TRAC⁻/β2M⁻/CD70⁻), BCMA CAR⁺ T cells; or 4× KO (PD-1, CD70)(TRAC⁻/β2M⁻/PD1⁻/CD70⁻), BCMA CAR⁺ T cells.

FIG. 28B includes a graph showing tumor volume reduction in asubcutaneous MM.1S model following a tumor cell re-challenge. Mice fromFIG. 28A were re-challenged with a second inoculation of MM.1S cells onday 45 and tumor volume was assessed over time.

FIG. 29 includes graphs showing the number of human CD45⁺ 2× KO(TRAC⁻/β2M⁻), BCMA CAR⁺ T cells; human CD45⁺ 3× KO (PD-1)(TRAC⁻/β2M⁻/PD1⁻), BCMA CAR⁺ T cells; human CD45⁺ 3× KO (CD70)(TRAC⁻/β2M⁻/CD70⁻), BCMA CAR⁺ T cells; and human CD45⁺ 4× KO (PD-1,CD70) (TRAC⁻/β2M⁻/PD1⁻/CD70⁻), BCMA CAR⁺ T cells 1 week (right graph), 2weeks (middle graph), and 3 weeks (left graph) post dosing.

FIG. 30 includes graphs showing the results from an experiment designedto assess tumor volume reduction in a subcutaneous RPMI-8226 tumorxenograft model exposed to: TRAC⁻/β2M/-anti-BCMA CAR⁺ T cells (2× KO,BCMA CAR⁺ T cells); TRAC⁻/β2M⁻/PD1⁻/anti-BCMA CAR⁺ T cells (3× KO(PD-1), BCMA CAR⁺ T cells); TRAC⁻/β2M⁻/CD70⁻/anti-BCMA CAR⁺ T cells (3×KO (CD70), BCMA CAR⁺ T cells); or TRAC⁻/β2M⁻/PD1⁻/CD70⁻/anti-BCMA CAR⁺ Tcells (4× KO (PD-1, CD70), BCMA CAR⁺ T cells), at doses of 1×10⁵, 3×10⁵,1×10⁶, or 3×10⁶ cells/mouse.

FIG. 31 includes a graph showing that TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ Tcell maintain cytokine-dependent proliferation.

FIG. 32 shows cytokine-dependent growth of theTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells.

FIG. 33 includes a graph showing 4× KO (TRAC⁻/β2M⁻/PD-1⁻/CD70⁻), BCMACAR⁺ T cells maintain cytokine dependency.

FIG. 34 includes a graph showing enhanced cytokine (IL-2) release by 3×KO (TRAC−/β2M−/CD70−) anti-CD70 CAR+ T cells compared to 2× KO(TRAC−/β2M−) anti-CD70 CAR+ T cells when co-cultured with A498 kidneycancer cells at various ratios for 24 hours.

FIG. 35 includes a graph showing robust cell killing of A498 cells byanti-CD70 CAR T cells (2× KO (TRAC⁻/β2M⁻), CD70 CAR+; 3× KO (PD-1)(TRAC⁻/β2M⁻/PD-1⁻), CD70 CAR+; and 4× KO (TRAC⁻/β2M⁻/PD-1⁻/CD70⁻) CD70CAR⁺) relative to TCR+ T cells. T cells were co-cultured with A498 cellsat various ratios for 24 hours and percentage of cell lysis wasmeasured.

FIG. 36 includes a graph showing highest cell kill activity in A498kidney cancer cells using quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR+ T cells (4× KO, CD70 CAR+) andtriple knockout TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR+ T cells (3× KO (CD70),CD70 CAR+), relative to double knockout TRAC⁻/β2M⁻/anti-CD70 CAR⁺ (i.e.,2× KO, CD70 CAR⁺) T cells and triple knockout TRAC⁻/β2M⁻/PD-1⁻/anti-CD70CAR⁺ (i.e., 3× KO (PD-1), CD70 CAR⁺) T cells. A CAR T cell:A498 cellratio of 0.25:1 was used. Percentage of cell lysis of A498 cells wasmeasured 24 hours after co-culture.

FIGS. 37A and 37B include graphs showing that quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells (4× KO, CD70 CAR+) andtriple knockout TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells (3× KO (CD70),CD70 CAR+) secrete the highest levels of cytokines IFN-gamma (FIG. 37A)and IL-2 (FIG. 37B), relative to double knockout TRAC⁻/β2M⁻/anti-CD70CAR⁺ T cells (2× KO, CD70 CAR+) and triple knockoutTRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ T cells (3× KO (PD-1), CD70 CAR+). A CART cell:A498 cell ratio of 0.25:1 was used. IFN-gamma and IL-2 secretionwas measured 24 hours are co-culture.

FIG. 38 includes a graph showing that knocking out CD70 in anti-CD70 CART cells (3× KO (CD70) (TRAC⁻/β2M⁻/CD70⁻), CD70 CAR+; 3× KO (PD-1)(TRAC⁻/β2M⁻/PD1⁻), CD70 CAR+; and 4× KO (TRAC⁻/β2M⁻/CD70⁻/PD-1⁻), CD70CAR+) decreased levels of PD-1 expression in CD4+ T cells relative toanti-CD70 CAR T cells expressing endogenous CD70 (2× KO (TRAC⁻/β2M⁻)CD70 CAR+).

FIG. 39A and FIG. 39B include graphs showing that knocking out CD70 inanti-CD70 CAR T cells (3× KO (CD70) (TRAC⁻/β2M⁻/CD70⁻), CD70 CAR+; 3× KO(PD-1) (TRAC⁻/β2M⁻/PD1⁻), CD70 CAR+; and 4× KO (TRAC⁻/β2M⁻/CD70⁻/PD-1⁻),CD70 CAR+) decreased levels of exhaustion marker LAG3 in CD8+ T cells(FIG. 39A) and CD4+ T cells (FIG. 39B) relative to anti-CD70 CAR T cellsexpressing endogenous CD70 (2× KO (TRAC⁻/β2M⁻) CD70 CAR+).

FIG. 40A includes graphs showing relative CD70 expression in fivedifferent cancer cell lines (left panel) and relative CD70 expression inthree different cancel cell lines (right panel). FIG. 40B includesgraphs showing relative CD70 expression in nine different cancer celllines. FIGS. 40C-40D include graphs showing highest cell kill activityin ACHN (ATCC® CRL-1611™) kidney cancer cells (which express low levelsof CD70) using quadruple knockout TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ Tcells (4× KO, CD70 CAR+) and triple knockout TRAC⁻/β2M⁻/CD70⁻/anti-CD70CAR⁺ T cells (3× KO (CD70), CD70 CAR+), relative to double knockoutTRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cells (2× KO, CD70 CAR+) and triple knockoutTRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ T cells (3× KO (PD-1), CD70 CAR+). A CART cell:ACHN cell ratio of 0.5:1 was used in FIG. 40C and a CAR Tcell:ACHN cell ratio of 0.25:1 was used in FIG. 40D. FIG. 40E and FIG.40F include graphs showing cell kill activity using quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells (FIG. 40E) and tripleknockout TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells (FIG. 40F) againstadditional solid tumor cell lines with varying levels of CD70 expression(4:1, 1:1, or 0.25:1 effector:target cell ratio). FIG. 40G includes agraph showing cell kill activity using the triple knockoutTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells against solid tumor cell linesafter a co-culture period of 24 hours or 96 hours. FIGS. 40H-40J includegraphs showing cell kill activity using the triple knockoutTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells (3KO (CD70), CD70 CAR+) againstCD70-deficient chronic myelogenous leukemia (K562) cells (FIG. 40H),CD70-expressing multiple myeloma (MM.1S) cells (FIG. 40I), andCD70-expressing T cell lymphoma (HuT78) cells (FIG. 40J) at variouseffector:target ratios.

FIG. 41A and FIG. 41B include graphs showing that quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells (4× KO, CD70 CAR+) andtriple knockout TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells (3× KO (CD70),CD70 CAR+) secrete the highest levels of cytokines IFN-gamma (FIG. 41A)and IL-2 (FIG. 41B), relative to double knockout TRAC⁻/β2M⁻/anti-CD70CAR⁺ T cells (2× KO, CD70 CAR+) and triple knockoutTRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ T cells (3× KO (PD-1), CD70 CAR+). A CART cell:ACHN cell ratio of 0.25:1 was used.

FIG. 42A includes a graph showing results from an experiment designed toassess tumor volume reduction in a human ovarian tumor xenograft model(e.g., SKOV-3 tumor cells) exposed to 3× KO (TRAC−/B2M−/CD70−) anti-CD70CAR T cells. FIG. 42B includes a graph showing results from anexperiment designed to assess tumor volume reduction in a humannon-small cell lung tumor xenograft model (e.g., NCI-H1975 tumor cells)exposed to 3× KO (TRAC−/B2M−/CD70−) anti-CD70 CAR T cells. FIG. 42Cincludes a graph showing results from an experiment designed to assesstumor volume reduction in a human pancreatic tumor xenograft model(e.g., Hs766T tumor cells) exposed to 3× KO (TRAC−/B2M−/CD70−) anti-CD70CAR T cells. FIG. 42D includes graphs showing results from an experimentdesigned to assess tumor volume reduction in a human T-cell lymphomaxenograft model (e.g., HuT78 tumor cells) exposed to 3× KO(TRAC−/B2M−/CD70−) anti-CD70 CAR T cells. Tumor volumes of individualmice (left) and mean tumor volumes (right) are shown.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the discovery thatdisrupting the CD70 gene in immune cells engineered to express anantigen targeting moiety (e.g., a CAR) enhances several characteristicsimportant for cell-based immunotherapy, including anti-tumor efficacy.Specifically, such engineered immune cells showed unexpected superiorfeatures, including extended proliferation and in vivo persistenceresulting in long-term, enhanced anti-tumor efficacy. Notably, theseunexpected features have been demonstrated with targeting moietiesspecific for various antigens, including BCMA, CD19, CD33 and CD70.

As demonstrated herein, disrupting the CD70 gene resulted in maintenanceof cytotoxicity of immune cells engineered to express an antigentargeting moiety after multiple rounds of challenges by cancer cells invitro. Without wishing to be bound by theory, this maintenance ofcytotoxicity indicates disrupting the CD70 gene makes the engineeredimmune cells resistant to exhaustion and may result in cells that livelonger.

It was also found that disrupting the CD70 gene in immune cellsengineered to express an antigen targeting moiety enhanced anti-tumorefficacy against large tumors and induced a durable anti-cancer memoryresponse. Specifically, the anti-cancer memory response prevented tumorgrowth upon re-challenge. Further, it has been demonstrated disruptingthe CD70 gene results in enhanced cytotoxicity of immune cellsengineered to express an antigen targeting moiety at lower ratios ofengineered immune cells to target cells, indicating the potentialefficacy of low doses of engineered immune cells.

It has also been shown disruption of the CD70 gene enhances cellproliferation and in vivo persistence of engineered immune cells.Without wishing to be bound by theory, it is believed the superiorfeatures of the engineered immune cells described herein allow for moreconsistent cell populations, larger scale production due to the cells'ability to survive more cell division, and fewer starting cells requiredto produce the engineered cells. Such features may also prove beneficialin a clinical setting. For example, increased expansion and decreasedexhaustion indicates increased efficacy per dose and the ability toobtain efficacy with lower doses.

It has also been demonstrated that disrupting the CD70 gene in immunecells engineered to express an antigen targeting moiety maintainscytotoxicity against cancer cells expressing highly immune suppressivemolecules, i.e., PD-L1. Without wishing to be bound by theory, it isbelieved the internal negative signal of PD-1 expressed on immune cellswhen bound to PD-L1 expressed on cancer cells, is overcome by disruptingCD70.

Accordingly, provided herein are methods and compositions (e.g., cellcompositions) for the treatment of cancer, such as BCMA⁺, CD19⁺, CD33⁺,and CD70⁺ malignancies, involving the use of the engineered immune cellswith increased efficacy and persistence.

CD70 Gene Edit

Cluster of Differentiation 70 (CD70) is a member of the tumor necrosisfactor superfamily and its expression is restricted to activated T and Blymphocytes and mature dendritic cells. CD70 is implicated in tumor celland regulatory T cell survival through interaction with its ligand,CD27. CD70 and its receptor CD27 have multiple roles in immune functionin multiple cell types including T cells (activated and T regs), and Bcells. It is unclear exactly how CD70 functions in all of these celltypes to control functions such as apoptosis, with publicationsindicating contradicting roles. For example, it has been reported thatCD70 induces apoptosis or survival of T cells depending on the antigenicload (Wensveen, F., et al. J. Immunol, Vol 188: 4256-4267, 2012).

While CAR T cells have proved to be an effective immunotherapeutic,various challenges remain. For example, over time CAR T cells becomeexhausted and become ineffective in vivo. With regards to manufacturing,it takes significant time to produce enough cells to dose a patient. Toaddress these limitations, the present disclosure provides CAR T cellsthat have been engineered to disrupt endogenous CD70 expression while atthe same time expressing an antigen targeting moiety (e.g., an scFv).

Surprisingly, the present disclosure shows disrupting the CD70 geneenables increased CAR T health and function (e.g., extendedproliferation, reduced exhaustion) regardless of the antigen beingtargeted by the scFv in the CAR T. This applies even to antigensexpressed on T cells such as CD33 and CD70 where the effects of thedisrupted CD70 gene retain CAR T function even where fratricide may beexpected. That is, these CD70 knockout cells (e.g., in which the CD70gene has been edited using CRISPR/Cas9 gene editing technology),independent of the CAR insertion, exhibit continued, steady cell growth,relative to unmodified T cells (or edited T cells that express CD70) andexpress lower levels of exhaustion markers, such as LAGS. The CAR Tcells of the present disclosure, may include any antibody (includingwhole antibodies and antibody fragments) or other molecule (e.g.,receptor or ligand) that specifically binds to a cancer antigen to guidethe CAR T cell to a cancer cell. In some embodiments, the antibody is ananti-CD70 antibody (e.g., an anti-CD70 scFv). In other embodiments, theantibody is an anti-CD19 antibody (e.g., an anti-CD19 scFv). In yetother embodiments, the antibody is an anti-BCMA antibody (e.g., ananti-BCMA scFv). In other embodiments, the antibody is an anti-CD33antibody (e.g.,, an anti-CD33 scFv). Other cancer antigens areencompassed by the present disclosure.

It should be understood that gene disruption encompasses genemodification through gene editing (e.g., using CRISPR/Cas gene editingto insert or delete one or more nucleotides). In some embodiments, adisrupted gene is a gene that does not encode functional protein. Insome embodiments, a cell that comprises a disrupted gene does notexpress (e.g., at the cell surface) a detectable level (e.g. byantibody, e.g., by flow cytometry) of the protein encoded by the gene. Acell that does not express a detectable level of the protein may bereferred to as a knockout cell. For example, a cell having a CD70 geneedit may be considered a CD70 knockout cell if CD70 protein cannot bedetected at the cell surface using an antibody that specifically bindsCD70 protein.

Provided herein, in some embodiments, are populations of cells in whicha certain percentage of the cells has been edited (e.g., CD70 geneedited), resulting in a certain percentage of cells not expressing aparticular gene and/or protein. In some embodiments, at least 50% (e.g.,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 85%) of the cells of agene-edited population of cells are CD70 knockout cells. In someembodiments, at least 50% of the cells (e.g. T cells) of the populationdo not express detectable levels of CD70 protein. In some embodiments,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% of the cells of agene-edited population of cells may be CD70 knockout cells.

In some embodiments, 10%, 15%, 20%, 25%, 30%, 35% or 40% of theengineered T cells of a population do not express a detectable level ofCD70 surface protein. In some embodiments, the percent of engineered Tcells that do not express a detectable level of CD70 surface proteinincreases over time. Thus, in some embodiments, at least 50% of theengineered T cells of a population of engineered T cells does notexpress a detectable level of CD70 surface protein. For example, atleast 55%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% of the engineered T cells of apopulation may not express a detectable level of CD70 surface protein.In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%,60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%- 90%, 70%-80%,80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a populationdoes not express a detectable level of CD70 surface protein.

Non-limiting examples of modified and unmodified CD70 gRNA sequencesthat may be used as provided herein to create a genomic alteration(e.g., disruption, e.g., deletion, insertion, substitution) in the CD70gene are listed in Table 5 (e.g., SEQ ID NOS: 23-29 and 33-39). OthergRNA sequences may be designed using the CD70 gene sequence located onChromosome 19 (GRCh38 coordinates: Chromosome 19: 6,583,183-6,604,103;Ensembl: ENSG00000125726). In certain embodiments, gRNAs targeting theCD70 genomic region create Indels (e.g.: insertions, deletions orsubstitutions) in, or around, the CD70 gene disrupting expression of theCD70 mRNA and/or protein.

In some embodiments, a ribonucleoprotein particle (RNP) containing anRNA-guided nuclease (e.g., a Cas nuclease, such as a Cas9 nuclease) anda gRNA targeting the CD70 gene (or any other gene of interest) aredelivered to T cells (e.g., primary T cells). In other embodiments, theRNA-guided nuclease and gRNA are delivered separately to T cells. Aribonucleoprotein particle (RNP) is simply an RNA-guided nuclease (e.g.,Cas9) pre-complexed/complexed with (bound to) a gRNA.

In some embodiments, the gRNA targeting the CD70 gene is a syntheticmodified gRNA such as but not limited to any one of the gRNAs comprisingSEQ ID NO: 33-39. In some embodiments, the gRNA targeting the CD70 geneis a synthetic unmodified gRNA such as but not limited to any one of thegRNAs comprising SEQ ID NO: 23-29.

In some embodiments, gRNAs targeting the CD70 genomic region andRNA-guided nuclease create double stranded breaks in the CD70 gene.Repair of the break results in Indels in the CD70 gene wherein the CD70gene sequence may comprises a nucleotide sequence selected from thegroup consisting of: SEQ ID NOs: 129-134.

Multi-Gene Editing

The engineered T cells of the present disclosure, in some embodiments,include more than one disrupted gene (e.g.: more than one gene edit),for example, in more than one gene. For example, an engineered T cellmay comprise a disrupted CD70 gene, a disrupted T cell receptor alphachain constant region (TRAC) gene, a disrupted beta-2-microglobulin(β2M) gene, a disrupted programmed cell death-1 (PD-1 or PDCD1) gene, orany combination of two or more of the foregoing disrupted genes. In someembodiments, an engineered T cell comprises a disrupted TRAC gene, adisrupted β2M gene, and a disrupted CD70 gene. In some embodiments, anengineered T cell comprises a disrupted TRAC gene, a disrupted β2M gene,and a disrupted PD-1 gene. In some embodiments, an engineered T cellcomprises a disrupted TRAC gene, a disrupted β2M gene, a disrupted CD70gene and a disrupted PD-1 gene.

TRAC Gene Edit

In some embodiments, an engineered T cell comprises a disrupted TRACgene. This disruption leads to loss of function of the TCR and rendersthe engineered T cell non-alloreactive and suitable for allogeneictransplantation, minimizing the risk of graft versus host disease. Insome embodiments, expression of the endogenous TRAC gene is eliminatedto prevent a graft-versus-host response.

In some embodiments, a disruption in the TRAC gene expression is createdby knocking a chimeric antigen receptor (CAR) into the TRAC gene (e.g.,using an adeno-associated viral (AAV) vector and donor template). Insome embodiments, a disruption in the TRAC gene expression is createdwith a nuclease and gRNAs targeting the TRAC genomic region. In someembodiments, a genomic deletion in the TRAC gene is created by HDR,wherein a chimeric antigen receptor (CAR) replaces a segment of the TRACgene (e.g., using an adeno-associated viral (AAV) vector and donortemplate). In some embodiments, a disruption in the TRAC gene expressionis created with a nuclease and gRNAs targeting the TRAC genomic region,and knocking a chimeric antigen receptor (CAR) into the TRAC gene.

Non-limiting examples of modified and unmodified TRAC gRNA sequencesthat may be used as provided herein to create a genomic in the TRAC geneare listed in Table 7 (e.g., SEQ ID NOS: 30 and 40). See alsoInternational Application No. PCT/US2018/032334, filed May 11, 2018,incorporated herein by reference. Other gRNA sequences may be designedusing the TRAC gene sequence located on chromosome 14 (GRCh38:chromosome 14: 22,547,506-22,552,154;. Ensembl; ENSG00000277734). Insome embodiments, gRNAs targeting the TRAC genomic region and RNA-guidednuclease create breaks in the TRAC genomic region resulting Indels inthe TRAC gene disrupting expression of the mRNA or protein.

In some embodiments, at least 50% of the engineered T cells of apopulation do not express a detectable level of T cell receptor (TCR)surface protein. For example, at least 55%, at least 60%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%of the engineered T cells of a population may not express a detectablelevel of TCR surface protein. In some embodiments, 50%-100%, 50%-90%,50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%,70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of theengineered T cells of a population do not express a detectable level ofTCR surface protein.

In some embodiments, a ribonucleoprotein particle (RNP) containing anRNA-guided nuclease (e.g., a Cas nuclease, such as a Cas9 nuclease) anda gRNA targeting the TRAC gene (or any other gene of interest) aredelivered to T cells (e.g., primary T cells). In other embodiments, theRNA-guided nuclease and gRNA are delivered separately to T cells. Aribonucleoprotein particle (RNP) is simply an RNA-guided nuclease (e.g.,Cas9) pre-complexed/complexed with a gRNA.

In some embodiments, gRNAs and RNA-guided nuclease targeting the TRACgenomic region result Indels in the TRAC gene comprising a nucleotidesequence selected from the following sequences in Table 1:

TABLE 1 SEQ ID Sequence NO: AAGAGCAACAAATCTGACT 1AAGAGCAACAGTGCTGTGCCTGGAGCAACAAATCTGACT 2 AAGAGCAACAAATCTGACTAAGAGCAACAGTGCTGGAGCAACAAATCTGACT 3 AAGAGCAACAAATCTGACTAAGAGCAACAGTGCCTGGAGCAACAAATCTGACT 4 AAGAGCAACAAATCTGACTAAGAGCAACAGTGCTGACTAAGAGCAACAAATCTGACT 5AAGAGCAACAGTGCTGTGGGCCTGGAGCAACAAATCTGACT 6 AAGAGCAACAAATCTGACTAAGAGCAACAGTGCTGGCCTGGAGCAACAAATCTGACT 7 AAGAGCAACAAATCTGACTAAGAGCAACAGTGCTGTGTGCCTGGAGCAACAAATCTGACT 8 AAGAGCAACAAATCTGACT

In some embodiments, an engineered T cell comprises a deletion in theTRAC gene relative to unmodified T cells. In some embodiments, anengineered T cell comprises a deletion of 15-30 base pairs in the TRACgene relative to unmodified T cells. In some embodiments, an engineeredT cell comprises a deletion of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 base pairs in the TRAC gene relative tounmodified T cells. In some embodiments, an engineered T cell comprisesa deletion of more than 30 base pairs in the TRAC gene relative tounmodified T cells. In some embodiments, an engineered T cell comprisesa deletion of 20 base pairs in the TRAC gene relative to unmodified Tcells. In some embodiments, an engineered T cell comprises a deletion ofSEQ ID NO: 86 in the TRAC gene relative to unmodified T cells. In someembodiments, an engineered T cell comprises a deletion comprising SEQ IDNO: 86 in the TRAC gene relative to unmodified T cells. In someembodiments, an engineered T cell comprises a deletion of SEQ ID NO: 118in the TRAC gene relative to unmodified T cells. In some embodiments, anengineered T cell comprises a deletion comprising SEQ ID NO: 118 in theTRAC gene relative to unmodified T cells.

β2M Gene Edit

In some embodiments, an engineered T cell comprises a disrupted β2Mgene. β2M is a common (invariant) component of MHC I complexes.Disrupting its expression by gene editing will prevent host versustherapeutic allogeneic T cells responses leading to increased allogeneicT cell persistence. In some embodiments, expression of the endogenousβ2M gene is eliminated to prevent a host-versus-graft response.

Non-limiting examples of modified and unmodified β2M gRNA sequences thatmay be used as provided herein to create a genomic deletion in the β2Mgene are listed in Table 7 (e.g., SEQ ID NOS: 31 and 41). See alsoInternational Application No. PCT/US2018/032334, filed May 11, 2018,incorporated herein by reference. Other gRNA sequences may be designedusing the β2M gene sequence located on Chromosome 15 (GRCh38coordinates: Chromosome 15: 44,711,477-44,718,877; Ensembl:ENSG00000166710).

In some embodiments, gRNAs targeting the β2M genomic region andRNA-guided nuclease create breaks in the β2M genomic region resulting inIndels in the β2M gene disrupting expression of the mRNA or protein.

In some embodiments, at least 50% of the engineered T cells of apopulation do not express a detectable level of β2M surface protein. Forexample, at least 55%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% of the engineeredT cells of a population may not express a detectable level of β2Msurface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%,50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%,70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered Tcells of a population do not express a detectable level of β2M surfaceprotein.

In some embodiments, less than 50% of the engineered T cells of apopulation of cells express a detectable level of β2M surface protein.In some embodiments, less than 30% of the engineered T cells of apopulation of cells express a detectable level of β2M surface protein.For example, less than 50%, less than 30%, less than 25%, less than 20%,less than 15%, less than 10%, or less than 5% of the engineered T cellsof a population of cells express a detectable level of β2M surfaceprotein. In some embodiments, 40%-30%, 40%-20%, 40% -10%, 40%-5%,30%-20%, 30%-10%, 30%-5%, 20%-10%, 20%-5%, or 10%-5% of the engineered Tcells of a population of cells express a detectable level of β2M surfaceprotein.

In some embodiments, a ribonucleoprotein particle (RNP) containing anRNA-guided nuclease (e.g., a Cas nuclease, such as a Cas9 nuclease) anda gRNA targeting the B2M gene (or any other gene of interest) aredelivered to T cells (e.g., primary T cells). In other embodiments, theRNA-guided nuclease and gRNA are delivered separately to T cells. Aribonucleoprotein particle (RNP) is simply a RNA-guided nuclease (e.g.,Cas9) pre-complexed/complexed with a gRNA.

In some embodiments, an edited β2M gene comprises a nucleotide sequenceselected from the following sequences in Table 2.

TABLE 2 SEQ ID Sequences NO:CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGCCTGGA  9GGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCGCCTGGAG 10GCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGAGGCT 11ATCCAGCGTGAGTCTCTCCTACCCTCCCGCTCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGATAGC 12CTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTCGTGGCCTTAGCTGTGCTCGCGCTATCCAGCGTGAGTCTCTCCT 13 ACCCTCCCGCTCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGTGGCCT 14GGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT

PD-1 Gene Edit

PD-1 is an immune checkpoint molecule that is upregulated in activated Tcells and serves to dampen or stop T cell responses. Disrupting PD-1 bygene editing could lead to more persistent and/or potent therapeutic Tcell responses and/or reduce immune suppression in a subject. In someembodiments, an engineered T cell comprises a disrupted PD-1 gene. Insome embodiments, expression of the endogenous PD-1 gene is eliminatedto enhance anti-tumor efficacy of the CAR T cells of the presentdisclosure.

Non-limiting examples of modified and unmodified PD-1 gRNA sequencesthat may be used as provided herein to create a genomic deletion in thePD-1 gene are listed in Table 5 (e.g., SEQ ID NOS: 32 and 42). See alsoInternational Application No. PCT/US2018/032334, filed May 11, 2018,incorporated herein by reference. Other gRNA sequences may be designedusing the PD-1 gene sequence located on Chromosome 2 (GRCh38coordinates: Chromosome 2: 241,849,881-241,858,908; Ensembl:ENSG00000188389).

In some embodiments, gRNAs targeting and RNA-guided nuclease the PD-1genomic region create breaks in the TRAC genomic region resulting inIndels in the PD-1 gene disrupting expression of the PD-1 mRNA orprotein.

In some embodiments, at least 50% of the engineered T cells of apopulation do not express a detectable level of PD-1 surface protein.For example, at least 55%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% of the engineeredT cells of a population may not express a detectable level of PD-1surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%,50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%,70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered Tcells of a population do not express a detectable level of PD-1 surfaceprotein.

In some embodiments, a ribonucleoprotein particle (RNP) containing anRNA-guided nuclease (e.g., a Cas nuclease, such as a Cas9 nuclease) anda gRNA targeting the PD-1 gene (or any other gene of interest) aredelivered to T cells (e.g., primary T cells). In other embodiments, theRNA-guided nuclease and gRNA are delivered separately to T cells. Aribonucleoprotein particle (RNP) is simply an RNA-guided nuclease (e.g.,Cas9) pre-complexed/complexed with a gRNA.

Cellular Phenotypes

In some embodiments, one or more gene edits within a population of cellsresults in a phenotype associated with changes in cellular proliferativecapacity, cellular exhaustion, cellular viability, cellular lysiscapability (e.g., increase cytokine production and/or release), or anycombination thereof.

In some embodiments, engineered T cells of a population comprise a CARthat includes an anti-CD70 scFv ectodomain. In some embodiments,engineered T cells of a population comprise a CAR that includes ananti-BCMA scFv ectodomain. In some embodiments, engineered T cells of apopulation comprise a CAR that includes an anti-CD19 scFv ectodomain. Insome embodiments, engineered T cells of a population comprise a CAR thatincludes an anti-CD33 scFv ectodomain. Any of the foregoing engineered Tcells may also comprise a disruption in one or more of the followinggenes: TRAC, β2M, PD-1, and/or CD70 (e.g., TRAC⁻/β2M⁻/CD70⁻;TRAC⁻/β2M⁻/PD-1⁻; or TRAC⁻/β2M⁻/PD-1⁻/CD70⁻).

In some embodiments, engineered T cells of the present disclosureexhibit increased cellular proliferative capacity relative to controlcells. In some embodiments, engineered T cells of the present disclosureexhibit at least 20% greater cellular proliferative capacity, relativeto control T cells. For example, engineered T cells (e.g.,TRAC⁻/β2M⁻/CD70⁻; TRAC⁻/β2M⁻/PD-1⁻; or TRAC⁻/β2M⁻/PD-1⁻/CD70⁻; with orwithout a CAR) may exhibit at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, or at least 90%greater cellular proliferative capacity, relative to control T cells. Insome embodiments, engineered T cells of the present disclosure exhibit20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 30%-100%,30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 40%-100%, 40%-90%, 40%-80%,40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, or50%-60% greater cellular proliferative capacity, relative to control Tcells. Methods of measuring cell proliferation are known to those ofskill in the art and described herein.

In some embodiments, engineered T cells of the present disclosureexhibit reduced exhaustion, relative to control T cells. For example,the engineered T cells may express reduced levels of LAG3 (or otherexhaustion markers), relative to control T cells. In some embodiments,the levels of LAG3 expression are reduced by at least 20%, relative tocontrol T cells. For example, the levels of LAG3 expression may bereduced by at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, or at least 90%, relative tocontrol T cells. In some embodiments, the levels of LAG3 expression arereduced by 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%,30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 40%-100%,40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%,50%-70%, or 50%-60%, relative to control T cells. In some embodiments,reduced exhaustion is determined by measuring decreased surfaceexpression of exhaustion markers, including TIGIT, PD-1, LAG-3 orcombinations thereof. Methods for measuring surface expression are knownto those of skill in the art and described herein.

In some embodiments, engineered T cells of the present disclosureexhibit increased cellular viability relative to control cells. In someembodiments, engineered T cells of the present disclosure exhibit an atleast 20% increase in cellular viability, relative to control cells. Forexample, engineered T cells of the present disclosure may exhibit atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, or at least 90% increase in cellular viability,relative to control cells. In some embodiments, engineered T cells ofthe present disclosure exhibit a 20%-100%, 20%-90%, 20%-80%, 20%-70%,20%-60%, 20%-50%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%,40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%,50%-90%, 50%-80%, 50%-70%, or 50%-60% increase in cellular viability,relative to control cells. Methods of measuring cell viability are knownto those of skill in the art and described herein.

In some embodiments, engineered T cells of the present disclosureexhibit increased cellular lysis capability relative to control cells.In some embodiments, engineered T cells of the present disclosureexhibit an at least 20% increase in cellular lysis capability (kill atleast 20% more target cells), relative to control cells. For example,engineered T cells of the present disclosure may exhibit an at least atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, or at least 90% increase in cellular lysiscapability, relative to control cells. In some embodiments, engineered Tcells of the present disclosure exhibit a 20%-100%, 20%-90%, 20%-80%,20%-70%, 20%-60%, 20%-50%, 30%-100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%,30%-50%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%,50%-100%, 50%-90%, 50%-80%, 50%-70%, or 50%-60% increase in cellularlysis capability, relative to control cells.

In some embodiments, engineered T cells of the present disclosureexhibit increased cytokine secretion relative to control cells. Forexample, in some embodiments the level of cytokines (e.g., IL-2 and/orIFN-gamma) secreted by the engineered T cells is at least 2-fold (e.g.,at least 3-fold, at least 4-fold, or at least 5-fold) greater than thelevel of cytokines secreted by control T cells.

Control T cells, in some embodiments, are engineered T cells (e.g., geneedited T cells) that express endogenous CD70 protein (CD70 normallyexpressed by T cells). In some embodiments, control T cells areengineered T cells that express endogenous CD70 protein and comprise aTRAC gene disrupted by insertion of a nucleic acid encoding a CAR (e.g.,an anti-CD70 CAR or anti-BCMA CAR), a disrupted β2M gene, a disruptedPD-1 gene, or any combination of the foregoing disrupted genes. In someembodiments, control T cells are unedited T cells.

Surprisingly, the multi-gene edited CAR T cells of the presentdisclosure (e.g., TRAC⁻/β2M⁻/PD-1⁻/CD70⁻ cells) maintain cytotoxicity(ability to kill cancer cells), following multiple challenges (alsoreferred to as rechallenges(s)) with cancer cells. In some embodiments,the engineered T cells maintain cytotoxicity following at least 1rechallenge with a target cell, wherein the target cell expresses anantigen recognized by the CAR T cells. In some embodiments, theengineered T cells maintain cytotoxicity following at least 2rechallenges with a target cell, wherein the target cell expresses anantigen recognized by the CAR T cells. In some embodiments, theengineered T cells maintain cytotoxicity following at least 1rechallenge with a cancer cell. In some embodiments, the engineered Tcells maintain cytotoxicity following at least 2 rechallenges with acancer cell. In some embodiments, the engineered T cells maintaincytotoxicity following 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rechallengeswith a target cell, wherein the target cell expresses an antigenrecognized by the CAR T cells. In some embodiments, the engineered Tcells maintain cytotoxicity following 2, 3, 4, 5, 6, 7, 8, 9, or 10rechallenges with a target cell, wherein the target cell expresses anantigen recognized by the CAR T cells. In some embodiments, theengineered T cells maintain cytotoxicity following 2, 3, 4, 5, 6, 7, 8,9, or 10 rechallenges with a cancer cell. In some embodiments, theengineered T cells maintain cytotoxicity following 10 or morerechallenges with a a target cell, wherein the target cell expresses anantigen recognized by the CAR T cells. In some embodiments, theengineered T cells maintain cytotoxicity following 10 or morerechallenges with a cancer cell. In some embodiments, the engineered Tcells express a CAR specific for CD70 and the target cell (e.g., cancercell) expresses CD70. In some embodiments, the engineered T cellsexpress a CAR specific for CD19 and the target cell (e.g., cancer cell)expresses CD19. In some embodiments, the engineered T cells express aCAR specific for CD33 and the target cell (e.g., cancer cell) expressesCD33. In some embodiments, the engineered T cells express a CAR specificfor BCMA and the target cell (e.g., cancer cell) expresses BCMA.

Gene Editing Methods

Gene editing (including genomic editing) is a type of geneticengineering in which nucleotide(s)/nucleic acid(s) is/are inserted,deleted, and/or substituted in a DNA sequence, such as in the genome ofa targeted cell. Targeted gene editing enables insertion, deletion,and/or substitution at pre-selected sites in the genome of a targetedcell (e.g., in a targeted gene or targeted DNA sequence). When asequence of an endogenous gene is edited, for example by deletion,insertion or substitution of nucleotide(s)/nucleic acid(s), theendogenous gene comprising the affected sequence may be knocked-out orknocked-down due to the sequence alteration. Therefore, targeted editingmay be used to disrupt endogenous gene expression. “Targetedintegration” refers to a process involving insertion of one or moreexogenous sequences, with or without deletion of an endogenous sequenceat the insertion site. Targeted integration can result from targetedgene editing when a donor template containing an exogenous sequence ispresent. As used herein, a “disrupted gene” refers to a gene comprisingan insertion, deletion or substitution relative to an endogenous genesuch that expression of a functional protein from the endogenous gene isreduced or inhibited. As used herein, “disrupting a gene” refers to amethod of inserting, deleting or substituting at least onenucleotide/nucleic acid in an endogenous gene such that expression of afunctional protein from the endogenous gene is reduced or inhibited.Methods of disrupting a gene are known to those of skill in the art anddescribed herein.

Targeted editing can be achieved either through a nuclease-independentapproach, or through a nuclease-dependent approach. In thenuclease-independent targeted editing approach, homologous recombinationis guided by homologous sequences flanking an exogenous polynucleotideto be introduced into an endogenous sequence through the enzymaticmachinery of the host cell. The exogenous polynucleotide may introducedeletions, insertions or replacement of nucleotides in the endogenoussequence.

Alternatively, the nuclease-dependent approach can achieve targetedediting with higher frequency through the specific introduction ofdouble strand breaks (DSBs) by specific rare-cutting nucleases (e.g.,endonucleases). Such nuclease-dependent targeted editing also utilizesDNA repair mechanisms, for example, non-homologous end joining (NHEJ),which occurs in response to DSBs. DNA repair by NHEJ often leads torandom insertions or deletions (indels) of a small number of endogenousnucleotides. In contrast to NHEJ mediated repair, repair can also occurby a homology directed repair (HDR). When a donor template containingexogenous genetic material flanked by a pair of homology arms ispresent, the exogenous genetic material can be introduced into thegenome by HDR, which results in targeted integration of the exogenousgenetic material.

Available endonucleases capable of introducing specific and targetedDSBs include, but not limited to, zinc-finger nucleases (ZFN),transcription activator-like effector nucleases (TALEN), and RNA-guidedCRISPR-Cas9 nuclease (CRISPR/Cas9; Clustered Regular Interspaced ShortPalindromic Repeats Associated 9). Additionally, DICE (dual integrasecassette exchange) system utilizing phiC31 and Bxb1 integrases may alsobe used for targeted integration.

ZFNs are targeted nucleases comprising a nuclease fused to a zinc fingerDNA binding domain (ZFBD), which is a polypeptide domain that binds DNAin a sequence-specific manner through one or more zinc fingers. A zincfinger is a domain of about 30 amino acids within the zinc fingerbinding domain whose structure is stabilized through coordination of azinc ion. Examples of zinc fingers include, but not limited to, C2H2zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zincfinger domain is a domain not occurring in nature whosedesign/composition results principally from rational criteria, e.g.,application of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data. See, for example, U.S. Pat. Nos. 6,140,081;6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO98/53060; WO 02/016536 and WO 03/016496. A selected zinc finger domainis a domain not found in nature whose production results primarily froman empirical process such as phage display, interaction trap or hybridselection. ZFNs are described in greater detail in U.S. Pat. Nos.7,888,121 and 7,972,854. The most recognized example of a ZFN is afusion of the Fokl nuclease with a zinc finger DNA binding domain.

A TALEN is a targeted nuclease comprising a nuclease fused to a TALeffector DNA binding domain. A “transcription activator-like effectorDNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNAbinding domain” is a polypeptide domain of TAL effector proteins that isresponsible for binding of the TAL effector protein to DNA. TAL effectorproteins are secreted by plant pathogens of the genus Xanthomonas duringinfection. These proteins enter the nucleus of the plant cell, bindeffector-specific DNA sequences via their DNA binding domain, andactivate gene transcription at these sequences via their transactivationdomains. TAL effector DNA binding domain specificity depends on aneffector-variable number of imperfect 34 amino acid repeats, whichcomprise polymorphisms at select repeat positions called repeatvariable-diresidues (RVD). TALENs are described in greater detail in USPatent Application No. 2011/0145940. The most recognized example of aTALEN in the art is a fusion polypeptide of the Fokl nuclease to a TALeffector DNA binding domain.

Additional examples of targeted nucleases suitable for use as providedherein include, but are not limited to, Bxbl, phiC31, R4, PhiBT1, andWβ/SPBc/TP901-1, whether used individually or in combination.

Other non-limiting examples of targeted nucleases includenaturally-occurring and recombinant nucleases, e.g., CRISPR/Cas9,restriction endonucleases, meganucleases homing endonucleases, and thelike.

CRISPR-Cas9 Gene Editing

The CRISPR-Cas9 system is a naturally-occurring defense mechanism inprokaryotes that has been repurposed as an RNA-guided DNA-targetingplatform used for gene editing. It relies on the DNA nuclease Cas9, andtwo noncoding RNAs, crisprRNA (crRNA) and trans-activating RNA(tracrRNA), to target the cleavage of DNA. CRISPR is an abbreviation forClustered Regularly Interspaced Short Palindromic Repeats, a family ofDNA sequences found in the genomes of bacteria and archaea that containfragments of DNA (spacer DNA) with similarity to foreign DNA previouslyexposed to the cell, for example, by viruses that have infected orattacked the prokaryote. These fragments of DNA are used by theprokaryote to detect and destroy similar foreign DNA uponre-introduction, for example, from similar viruses during subsequentattacks. Transcription of the CRISPR locus results in the formation ofan RNA molecule comprising the spacer sequence, which associates withand targets Cas (CRISPR-associated) proteins able to recognize and cutthe foreign, exogenous DNA. Numerous types and classes of CRISPR/Cassystems have been described (see e.g., Koonin et al., (2017) Curr OpinMicrobiol 37:67-78).

crRNA drives sequence recognition and specificity of the CRISPR-Cas9complex through Watson-Crick base pairing typically with a 20 nucleotide(nt) sequence in the target DNA. Changing the sequence of the 5′ 20nt inthe crRNA allows targeting of the CRISPR-Cas9 complex to specific loci.The CRISPR-Cas9 complex only binds DNA sequences that contain a sequencematch to the first 20 nt of the crRNA, single-guide RNA (sgRNA), if thetarget sequence is followed by a specific short DNA motif (with thesequence NGG) referred to as a protospacer adjacent motif (PAM).

TracrRNA hybridizes with the 3′ end of crRNA to form an RNA-duplexstructure that is bound by the Cas9 endonuclease to form thecatalytically active CRISPR-Cas9 complex, which can then cleave thetarget DNA.

Once the CRISPR-Cas9 complex is bound to DNA at a target site, twoindependent nuclease domains within the Cas9 enzyme each cleave one ofthe DNA strands upstream of the PAM site, leaving a double-strand break(DSB) where both strands of the DNA terminate in a base pair (a bluntend).

After binding of CRISPR-Cas9 complex to DNA at a specific target siteand formation of the site-specific DSB, the next key step is repair ofthe DSB. Cells use two main DNA repair pathways to repair the DSB:non-homologous end-joining (NHEJ) and homology-directed repair (HDR).

NHEJ is a robust repair mechanism that appears highly active in themajority of cell types, including non-dividing cells. NHEJ iserror-prone and can often result in the removal or addition of betweenone and several hundred nucleotides at the site of the DSB, though suchmodifications are typically <20 nt. The resulting insertions anddeletions (indels) can disrupt coding or noncoding regions of genes.Alternatively, HDR uses a long stretch of homologous donor DNA, providedendogenously or exogenously, to repair the DSB with high fidelity. HDRis active only in dividing cells, and occurs at a relatively lowfrequency in most cell types. In many embodiments of the presentdisclosure, NHEJ is utilized as the repair operant.

In some embodiments, the Cas9 (CRISPR associated protein 9) endonucleaseis from Streptococcus pyogenes, although other Cas9 homologs may beused. It should be understood, that wild-type Cas9 may be used ormodified versions of Cas9 may be used (e.g., evolved versions of Cas9,or Cas9 orthologues or variants), as provided herein. In someembodiments, Cas9 may be substituted with another RNA-guidedendonuclease, such as Cpf1 (of a class II CRISPR/Cas system).

In some embodiments, the CRISPR/Cas system comprise components derivedfrom a Type-I, Type-II, or Type-III system. Updated classificationschemes for CRISPR/Cas loci define Class 1 and Class 2 CRISPR/Cassystems, having Types Ito V or VI (Makarova et al., (2015) Nat RevMicrobiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397).Class 2 CRISPR/Cas systems have single protein effectors. Cas proteinsof Types II, V, and VI are single-protein, RNA-guided endonucleases,herein called “Class 2 Cas nucleases.” Class 2 Cas nucleases include,for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9,and contains a RuvC-like nuclease domain.

In some embodiments, the Cas nuclease is from a Type-II CRISPR/Cassystem (e.g., a Cas9 protein from a CRISPR/Cas9 system). In someembodiments, the Cas nuclease is from a Class 2 CRISPR/Cas system (asingle-protein Cas nuclease such as a Cas9 protein or a Cpf1 protein).The Cas9 and Cpf1 family of proteins are enzymes with DNA endonucleaseactivity, and they can be directed to cleave a desired nucleic acidtarget by designing an appropriate guide RNA, as described furtherherein.

In some embodiments, a Cas nuclease may comprise more than one nucleasedomain. For example, a Cas9 nuclease may comprise at least one RuvC-likenuclease domain (e.g. Cpf1) and at least one HNH-like nuclease domain(e.g. Cas9). In some embodiments, the Cas9 nuclease introduces a DSB inthe target sequence. In some embodiments, the Cas9 nuclease is modifiedto contain only one functional nuclease domain. For example, the Cas9nuclease is modified such that one of the nuclease domains is mutated orfully or partially deleted to reduce its nucleic acid cleavage activity.In some embodiments, the Cas9 nuclease is modified to contain nofunctional RuvC-like nuclease domain. In other embodiments, the Cas9nuclease is modified to contain no functional HNH-like nuclease domain.In some embodiments in which only one of the nuclease domains isfunctional, the Cas9 nuclease is a nickase that is capable ofintroducing a single-stranded break (a “nick”) into the target sequence.In some embodiments, a conserved amino acid within a Cas9 nucleasenuclease domain is substituted to reduce or alter a nuclease activity.In some embodiments, the Cas nuclease nickase comprises an amino acidsubstitution in the RuvC-like nuclease domain. Exemplary amino acidsubstitutions in the RuvC-like nuclease domain include D10A (based onthe S. pyogenes Cas9 nuclease). In some embodiments, the nickasecomprises an amino acid substitution in the HNH-like nuclease domain.Exemplary amino acid substitutions in the HNH-like nuclease domaininclude E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenesCas9 nuclease).

In some embodiments, the Cas nuclease is from a Type-I CRISPR/Cassystem. In some embodiments, the Cas nuclease is a component of theCascade complex of a Type-I CRISPR/Cas system. For example, the Casnuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease isderived from a Type-III CRISPR/Cas system. In some embodiments, the Casnuclease is derived from Type-IV CRISPR/Cas system. In some embodiments,the Cas nuclease is derived from a Type-V CRISPR/Cas system. In someembodiments, the Cas nuclease is derived from a Type-VI CRISPR/Cassystem.

Guide RNAs

The present disclosure provides a genome-targeting nucleic acid that candirect the activities of an associated polypeptide (e.g., asite-directed polypeptide) to a specific target sequence within a targetnucleic acid. The genome-targeting nucleic acid can be an RNA. Agenome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. Aguide RNA comprises at least a spacer sequence that hybridizes to atarget nucleic acid sequence of interest, and a CRISPR repeat sequence.In Type II systems, the gRNA also comprises a second RNA called thetracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence andtracrRNA sequence hybridize to each other to form a duplex. In the TypeV gRNA, the crRNA forms a duplex. In both systems, the duplex binds asite-directed polypeptide, such that the guide RNA and site-directpolypeptide form a complex. In some embodiments, the genome-targetingnucleic acid provides target specificity to the complex by virtue of itsassociation with the site-directed polypeptide. The genome-targetingnucleic acid thus directs the activity of the site-directed polypeptide.

As is understood by the person of ordinary skill in the art, each guideRNA is designed to include a spacer sequence complementary to itsgenomic target sequence. See Jinek et al., Science, 337, 816-821 (2012)and Deltcheva et al., Nature, 471, 602-607 (2011).

In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is adouble-molecule guide RNA. In some embodiments, the genome-targetingnucleic acid (e.g., gRNA) is a single-molecule guide RNA.

A double-molecule guide RNA comprises two strands of RNA. The firststrand comprises in the 5′ to 3′ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand comprises a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (referred to as a “sgRNA”) in a Type IIsystem comprises, in the 5′ to 3′ direction, an optional spacerextension sequence, a spacer sequence, a minimum

CRISPR repeat sequence, a single-molecule guide linker, a minimumtracrRNA sequence, a 3′ tracrRNA sequence and an optional tracrRNAextension sequence. The optional tracrRNA extension may compriseelements that contribute additional functionality (e.g., stability) tothe guide RNA. The single-molecule guide linker links the minimum CRISPRrepeat and the minimum tracrRNA sequence to form a hairpin structure.The optional tracrRNA extension comprises one or more hairpins.

A single-molecule guide RNA in a Type V system comprises, in the 5′ to3′ direction, a minimum CRISPR repeat sequence and a spacer sequence.

In some embodiments, the sgRNA comprises a 20 nucleotide spacer sequenceat the 5′ end of the sgRNA sequence. In some embodiments, the sgRNAcomprises a less than 20 nucleotide spacer sequence at the 5′ end of thesgRNA sequence. In some embodiments, the sgRNA comprises a more than 20nucleotide spacer sequence at the 5′ end of the sgRNA sequence. In someembodiments, the sgRNA comprises a variable length spacer sequence with17-30 nucleotides at the 5′ end of the sgRNA sequence (see Table 3).

In some embodiments, the sgRNA comprises comprise no uracil at the 3′end of the sgRNA sequence. In some embodiments, the sgRNA comprisescomprise one or more uracil at the 3′ end of the sgRNA sequence. Forexample, the sgRNA can comprise 1 uracil (U) at the 3′ end of the sgRNAsequence. The sgRNA can comprise 2 uracil (UU) at the 3′ end of thesgRNA sequence. The sgRNA can comprise 3 uracil (UUU) at the 3′ end ofthe sgRNA sequence. The sgRNA can comprise 4 uracil (UUUU) at the 3′ endof the sgRNA sequence. The sgRNA can comprise 5 uracil (UUUUU) at the 3′end of the sgRNA sequence. The sgRNA can comprise 6 uracil (UUUUUU) atthe 3′ end of the sgRNA sequence. The sgRNA can comprise 7 uracil(UUUUUUU) at the 3′ end of the sgRNA sequence. The sgRNA can comprise 8uracil (UUUUUUUU) at the 3′ end of the sgRNA sequence.

The sgRNA can be unmodified or modified. For example, modified sgRNAscan comprise one or more 2′-O-methyl phosphorothioate nucleotides.

TABLE 3 SEQ ID NO. sgRNA sequence 15nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccga gucggugcuuuu 16nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccga gucggugc 17n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcu₍₁₋₈)

By way of illustration, guide RNAs used in the CRISPR/Cas/Cpf1 system,or other smaller RNAs can be readily synthesized by chemical means, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach used for generating RNAs of greater length isto produce two or more molecules that are ligated together. Much longerRNAs, such as those encoding a Cas9 or Cpf1 endonuclease, are morereadily generated enzymatically. Various types of RNA modifications canbe introduced during or after chemical synthesis and/or enzymaticgeneration of RNAs, e.g., modifications that enhance stability, reducethe likelihood or degree of innate immune response, and/or enhance otherattributes, as described in the art.

In some embodiments, indel frequency (editing frequency) may bedetermined using a TIDE analysis, which can be used to identify highlyefficient gRNA molecules. In some embodiments, a highly efficient gRNAyields a gene editing frequency of higher than 80%. For example, a gRNAis considered to be highly efficient if it yields a gene editingfrequency of at least 80%, at least 85%, at least 90%, at least 95%, or100%.

In some embodiments, gene disruption may occur by deletion of a genomicsequence using two guide RNAs. Methods of using CRISPR-Cas gene editingtechnology to create a genomic deletion in a cell (e.g., to knock out agene in a cell) are known (Bauer D E et al. Vis. Exp. 2015; 95;e52118).

Spacer Sequence

In some embodiments, a gRNA comprises a spacer sequence. A spacersequence is a sequence (e.g., a 20 nucleotide sequence) that defines thetarget sequence (e.g., a DNA target sequences, such as a genomic targetsequence) of a target nucleic acid of interest. In some embodiments, thespacer sequence is 15 to 30 nucleotides. In some embodiments, the spacersequence is 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleotides. In some embodiments, a spacer sequence is 20nucleotides.

The “target sequence” is adjacent to a PAM sequence and is the sequencemodified by an RNA-guided nuclease (e.g., Cas9). The “target nucleicacid” is a double-stranded molecule: one strand comprises the targetsequence and is referred to as the “PAM strand,” and the othercomplementary strand is referred to as the “non-PAM strand.” One ofskill in the art recognizes that the gRNA spacer sequence hybridizes tothe reverse complement of the target sequence, which is located in thenon-PAM strand of the target nucleic acid of interest. Thus, the gRNAspacer sequence is the RNA equivalent of the target sequence. Forexample, if the target sequence is 5′-AGAGCAACAGTGCTGTGGCC-3′ (SEQ IDNO: 86), then the gRNA spacer sequence is 5′-AGAGCAACAGUGCUGUGGCC-3′(SEQ ID NO: 98). The spacer of a gRNA interacts with a target nucleicacid of interest in a sequence-specific manner via hybridization (i.e.,base pairing). The nucleotide sequence of the spacer thus variesdepending on the target sequence of the target nucleic acid of interest.

In a CRISPR/Cas system herein, the spacer sequence is designed tohybridize to a region of the target nucleic acid that is located 5′ of aPAM of the Cas9 enzyme used in the system. The spacer may perfectlymatch the target sequence or may have mismatches. Each Cas9 enzyme has aparticular PAM sequence that it recognizes in a target DNA. For example,S. pyogenes recognizes in a target nucleic acid a PAM that comprises thesequence 5′-NRG-3′, where R comprises either A or G, where N is anynucleotide and N is immediately 3′ of the target nucleic acid sequencetargeted by the spacer sequence.

In some embodiments, the target nucleic acid sequence comprises 20nucleotides. In some embodiments, the target nucleic acid comprises lessthan 20 nucleotides. In some embodiments, the target nucleic acidcomprises more than 20 nucleotides. In some embodiments, the targetnucleic acid comprises at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30 or more nucleotides. In some embodiments, the targetnucleic acid comprises at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30 or more nucleotides. In some embodiments, the targetnucleic acid sequence comprises 20 bases immediately 5′ of the firstnucleotide of the PAM. For example, in a sequence comprising5′-NNNNNNNNNNNNNNNNNNNNNRG-3′, the target nucleic acid comprises thesequence that corresponds to the Ns, wherein N is any nucleotide, andthe underlined NRG sequence is the S. pyogenes PAM.

Non-limiting examples of gRNAs that may be used as provided herein areprovided in PCT/IB2018/001619, filed May 11, 2018, herein incorporatedby this reference.

Methods of Making gRNAs

The gRNAs of the present disclosure are produced by a suitable meansavailable in the art, including but not limited to in vitrotranscription (IVT), synthetic and/or chemical synthesis methods, or acombination thereof. Enzymatic (IVT), solid-phase, liquid-phase,combined synthetic methods, small region synthesis, and ligation methodsare utilized. In one embodiment, the gRNAs are made using IVT enzymaticsynthesis methods. Methods of making polynucleotides by IVT are known inthe art and are described in International Application PCT/US2013/30062.Accordingly, the present disclosure also includes polynucleotides, e.g.,DNA, constructs and vectors are used to in vitro transcribe a gRNAdescribed herein.

In some embodiments, non-natural modified nucleobases are introducedinto polynucleotides, e.g., gRNA, during synthesis or post-synthesis. Incertain embodiments, modifications are on internucleoside linkages,purine or pyrimidine bases, or sugar. In some embodiments, amodification is introduced at the terminal of a polynucleotide; withchemical synthesis or with a polymerase enzyme. Examples of modifiednucleic acids and their synthesis are disclosed in PCT application No.PCT/US2012/058519. Synthesis of modified polynucleotides is alsodescribed in Verma and Eckstein, Annual Review of Biochemistry, vol. 76,99-134 (1998).

In some embodiments, enzymatic or chemical ligation methods are used toconjugate polynucleotides or their regions with different functionalmoieties, such as targeting or delivery agents, fluorescent labels,liquids, nanoparticles, etc. Conjugates of polynucleotides and modifiedpolynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol.1(3), 165-187 (1990).

Certain embodiments of the invention also provide nucleic acids, e.g.,vectors, encoding gRNAs described herein. In some embodiments, thenucleic acid is a DNA molecule. In other embodiments, the nucleic acidis an RNA molecule. In some embodiments, the nucleic acid comprises anucleotide sequence encoding a crRNA. In some embodiments, thenucleotide sequence encoding the crRNA comprises a spacer flanked by allor a portion of a repeat sequence from a naturally-occurring CRISPR/Cassystem. In some embodiments, the nucleic acid comprises a nucleotidesequence encoding a tracrRNA. In some embodiments, the crRNA and thetracrRNA is encoded by two separate nucleic acids. In other embodiments,the crRNA and the tracrRNA is encoded by a single nucleic acid. In someembodiments, the crRNA and the tracrRNA is encoded by opposite strandsof a single nucleic acid. In other embodiments, the crRNA and thetracrRNA is encoded by the same strand of a single nucleic acid.

In some embodiments, the gRNAs provided by the disclosure are chemicallysynthesized by any means described in the art (see e.g., WO/2005/01248).While chemical synthetic procedures are continually expanding,purifications of such RNAs by procedures such as high performance liquidchromatography (HPLC, which avoids the use of gels such as PAGE) tendsto become more challenging as polynucleotide lengths increasesignificantly beyond a hundred or so nucleotides. One approach used forgenerating RNAs of greater length is to produce two or more moleculesthat are ligated together.

In some embodiments, the gRNAs provided by the disclosure aresynthesized by enzymatic methods (e.g., in vitro transcription, IVT).

Various types of RNA modifications can be introduced during or afterchemical synthesis and/or enzymatic generation of RNAs, e.g.,modifications that enhance stability, reduce the likelihood or degree ofinnate immune response, and/or enhance other attributes, as described inthe art.

In certain embodiments, more than one guide RNA can be used with aCRISPR/Cas nuclease system. Each guide RNA may contain a differenttargeting sequence, such that the CRISPR/Cas system cleaves more thanone target nucleic acid. In some embodiments, one or more guide RNAs mayhave the same or differing properties such as activity or stabilitywithin the Cas9 RNP complex. Where more than one guide RNA is used, eachguide RNA can be encoded on the same or on different vectors. Thepromoters used to drive expression of the more than one guide RNA is thesame or different.

The guide RNA may target any sequence of interest via the targetingsequence (e.g., spacer sequence) of the crRNA. In some embodiments, thedegree of complementarity between the targeting sequence of the guideRNA and the target sequence on the target nucleic acid molecule is about60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In someembodiments, the targeting sequence of the guide RNA and the targetsequence on the target nucleic acid molecule is 100% complementary. Inother embodiments, the targeting sequence of the guide RNA and thetarget sequence on the target nucleic acid molecule may contain at leastone mismatch. For example, the targeting sequence of the guide RNA andthe target sequence on the target nucleic acid molecule may contain 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, thetargeting sequence of the guide RNA and the target sequence on thetarget nucleic acid molecule may contain 1-6 mismatches. In someembodiments, the targeting sequence of the guide RNA and the targetsequence on the target nucleic acid molecule may contain 5 or 6mismatches.

The length of the targeting sequence may depend on the CRISPR/Cas9system and components used. For example, different Cas9 proteins fromdifferent bacterial species have varying optimal targeting sequencelengths. Accordingly, the targeting sequence may comprise 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. Insome embodiments, the targeting sequence may comprise 18-24 nucleotidesin length. In some embodiments, the targeting sequence may comprise19-21 nucleotides in length. In some embodiments, the targeting sequencemay comprise 20 nucleotides in length.

In some embodiments of the present disclosure, a CRISPR/Cas nucleasesystem includes at least one guide RNA. In some embodiments, the guideRNA and the Cas protein may form a ribonucleoprotein (RNP), e.g., aCRISPR/Cas complex. The guide RNA may guide the Cas protein to a targetsequence on a target nucleic acid molecule (e.g., a genomic DNAmolecule), where the the Cas protein cleaves the target nucleic acid. Insome embodiments, the CRISPR/Cas complex is a Cpf1 /guide RNA complex.In some embodiments, the CRISPR complex is a Type-II CRISPR/Cas9complex. In some embodiments, the Cas protein is a Cas9 protein. In someembodiments, the CRISPR/Cas9 complex is a Cas9/guide RNA complex.

Delivery of guide RNA and Nuclease

In some embodiments, a gRNA and an RNA-guided nuclease are delivered toa cell separately, either simultaneously or sequentially. In someembodiments, a gRNA and an RNA-guided nuclease are delivered to a celltogether. In some embodiments, a gRNA and an RNA-guided nuclease arepre-complexed together to form a ribonucleoprotein (RNP).

RNPs are useful for gene editing, at least because they minimize therisk of promiscuous interactions in a nucleic acid-rich cellularenvironment and protect the RNA from degradation. Methods for formingRNPs are known in the art. In some embodiments, an RNP containing anRNA-guided nuclease (e.g., a Cas nuclease, such as a Cas9 nuclease) anda gRNA targeting a gene of interest is delivered a cell (e.g.: a Tcell). In some embodiments, an RNP is delivered to a T cell byelectroporation.

As used herein, a “TRAC targeting RNP” refers to a gRNA that targets theTRAC gene pre-complexed with an RNA-guided nuclease. As used herein, a“β2M targeting RNP” refers to a gRNA that targets the β2M genepre-complexed with an RNA-guided nuclease. As used herein, a “CD70targeting RNP” refers to a gRNA that targets the CD70 gene pre-complexedwith an RNA-guided nuclease. As used herein, a “PD-1 targeting RNP”refers to a gRNA that targets the PD-1 gene pre-complexed with anRNA-guided nuclease.

In some embodiments, a TRAC targeting RNP is delivered to a cell. Insome embodiments, a β2M targeting RNP is delivered to a cell. In someembodiments, a CD70 targeting RNP is delivered to a cell. In someembodiments, a PD-1 targeting RNP is delivered to a cell.

In some embodiments, more than one RNP is delivered to a cell. In someembodiments, more than on RNP is delivered to a cell separately. In someembodiments, more than one RNP is delivered to a cell simultaneously. Insome embodiments, at least one of the following RNPs is delivered to acell:

(i) a TRAC targeting RNP;

(ii) a β2M targeting RNP;

(iii) a CD70 targeting RNP; or

(iv) a PD-1 targeting RNP. In some embodiments, at least two of thefollowing RNPs are delivered to a cell:

(i) a TRAC targeting RNP;

(ii) a β2M targeting RNP;

(iii) a CD70 targeting RNP; or

(iv) a PD-1 targeting RNP.

In some embodiments, an RNA-guided nuclease is delivered to a cell in aDNA vector that expresses the RNA-guided nuclease, an RNA that encodesthe RNA-guided nuclease, or a protein. In some embodiments, a gRNAtargeting a gene is delivered to a cell as an RNA, or a DNA vector thatexpresses the gRNA.

Delivery of an RNA-guided nuclease, gRNA, and/or an RNP may be throughdirect injection or cell transfection using known methods, for example,electroporation or chemical transfection. Other cell transfectionmethods may be used.

Chimeric antigen receptor (CAR) T cells

A chimeric antigen receptor refers to an artificial immune cell receptorthat is engineered to recognize and bind to an antigen expressed bytumor cells. Generally, a CAR is designed for a T cell and is a chimeraof a signaling domain of the T-cell receptor (TCR) complex and anantigen-recognizing domain (e.g., a single chain fragment (scFv) of anantibody or other antibody fragment) (Enblad et al., Human Gene Therapy.2015; 26(8):498-505). A T cell that expresses a CAR is referred to as aCAR T cell. CARs have the ability to redirect T-cell specificity andreactivity toward a selected target in a non-MHC-restricted manner. Thenon-MHC-restricted antigen recognition gives T-cells expressing CARs theability to recognize an antigen independent of antigen processing, thusbypassing a major mechanism of tumor escape. Moreover, when expressed inT-cells, CARs advantageously do not dimerize with endogenous T-cellreceptor (TCR) alpha and beta chains. CARs are often referenced to bythe antigen they bind. For example, a “CD19 CAR”, a “CD70 CAR”, a “CD33CAR” and a “BCMA CAR” are CARs comprising antigen binding domains thatspecifically bind to CD19, CD70, CD33 or BCMA, respectively.Accordingly, such terms are interchangeable with anti-CD19 CAR,anti-CD70 CAR, anti-CD33 CAR and anti-BCMA CAR. It will be understood bythose of ordinary skill in the art that a CAR that specifically binds anantigen can be referred to with either terminology.

There are four generations of CARs, each of which contains differentcomponents. First generation CARs join an antibody-derived scFv to theCD3zeta or z) intracellular signaling domain of the T-cell receptorthrough hinge and transmembrane domains. Second generation CARsincorporate an additional domain, e.g., CD28, 4-1BB (41BB), or ICOS, tosupply a costimulatory signal. Third-generation CARs contain twocostimulatory domains fused with the TCR CD3ζ chain. Third-generationcostimulatory domains may include, e.g., a combination of CD3ζ, CD27,CD28, 4-1BB, ICOS, or OX40. CARs, in some embodiments, contain anectodomain, commonly derived from a single chain variable fragment(scFv), a hinge, a transmembrane domain, and an endodomain with one(first generation), two (second generation), or three (third generation)signaling domains derived from CD3Z and/or co-stimulatory molecules(Maude et al., Blood. 2015; 125(26):4017-4023; Kakarla and Gottschalk,Cancer J. 2014; 20(2):151-155).

CARs typically differ in their functional properties. The CD3ζ signalingdomain of the T-cell receptor, when engaged, will activate and induceproliferation of T-cells but can lead to anergy (a lack of reaction bythe body's defense mechanisms, resulting in direct induction ofperipheral lymphocyte tolerance). Lymphocytes are considered anergicwhen they fail to respond to a specific antigen. The addition of acostimulatory domain in second-generation CARs improved replicativecapacity and persistence of modified T-cells. Similar antitumor effectsare observed in vitro with CD28 or 4-1BB CARs, but preclinical in vivostudies suggest that 4-1BB CARs may produce superior proliferationand/or persistence. Clinical trials suggest that both of thesesecond-generation CARs are capable of inducing substantial T-cellproliferation in vivo, but CARs containing the 4-1BB costimulatorydomain appear to persist longer. Third generation CARs combine multiplesignaling domains (costimulatory) to augment potency.

In some embodiments, a chimeric antigen receptor is a first generationCAR. In other embodiments, a chimeric antigen receptor is a secondgeneration CAR. In yet other embodiments, a chimeric antigen receptor isa third generation CAR.

A CAR, in some embodiments, comprises an extracellular (ecto) domaincomprising an antigen binding domain (e.g., an antibody, such as anscFv), a transmembrane domain, and a cytoplasmic (endo) domain.

Ectodomain

The ectodomain is the region of the CAR that is exposed to theextracellular fluid and, in some embodiments, includes an antigenbinding domain, and optionally a signal peptide, a spacer domain, and/ora hinge domain. In some embodiments, the antigen binding domain is asingle-chain variable fragment (scFv) that includes the VL and VH ofimmunoglobulins connected with a short linker peptide. The linker, insome embodiments, includes hydrophilic residues with stretches ofglycine and serine for flexibility as well as stretches of glutamate andlysine for added solubility. A single-chain variable fragment (scFv) isnot actually a fragment of an antibody, but instead is a fusion proteinof the variable regions of the heavy (VH) and light chains (VL) ofimmunoglobulins, connected with a short linker peptide of ten to about25 amino acids. The linker is usually rich in glycine for flexibility,as well as serine or threonine for solubility, and can either connectthe N-terminus of the VH with the C-terminus of the VL, or vice versa.This protein retains the specificity of the original immunoglobulin,despite removal of the constant regions and the introduction of thelinker. In some embodiments, the scFv of the present disclosure ishumanized. In other embodiments, the scFv is fully human. In yet otherembodiments, the scFv is a chimera (e.g., of mouse and human sequence).

In some embodiments, the scFv is an anti-CD70 scFv (binds specificallyto CD70). Non-limiting examples of anti-CD70 scFv proteins that may beused as provided herein may include the amino acid sequence of SEQ IDNO: 48 or SEQ ID NO: 50.

In some embodiments, the scFv is an anti-BCMA scFv (binds specificallyto BCMA). Non-limiting examples of anti-BCMA scFv proteins that may beused as provided herein may include the amino acid sequence of SEQ IDNO: 59.

In some embodiments, the scFv is an anti-CD19 scFv (binds specificallyto CD19). Non-limiting examples of anti-CD19 scFv proteins that may beused as provided herein may include the amino acid sequence of SEQ IDNO: 151.

In some embodiments, the scFv is an anti-CD33 scFv (binds specificallyto CD33). Non-limiting examples of anti-CD33 scFv proteins that may beused as provided herein may include the amino acid sequence of SEQ IDNO: 137.

Other scFv proteins may be used.

The signal peptide can enhance the antigen specificity of CAR binding.Signal peptides can be derived from antibodies, such as, but not limitedto, CD8, as well as epitope tags such as, but not limited to, GST orFLAG. Examples of signal peptides include MLLLVTSLLLCELPHPAFLLIP (SEQ IDNO: 88) and MALPVTALLLPLALLLHAARP (SEQ ID NO: 89). Other signal peptidesmay be used.

In some embodiments, a spacer domain or hinge domain is located betweenan extracellular domain (comprising the antigen binding domain) and atransmembrane domain of a CAR, or between a cytoplasmic domain and atransmembrane domain of the CAR. A spacer domain is any oligopeptide orpolypeptide that functions to link the transmembrane domain to theextracellular domain and/or the cytoplasmic domain in the polypeptidechain. A hinge domain is any oligopeptide or polypeptide that functionsto provide flexibility to the CAR, or domains thereof, or to preventsteric hindrance of the CAR, or domains thereof. In some embodiments, aspacer domain or a hinge domain may comprise up to 300 amino acids(e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In someembodiments, one or more spacer domain(s) may be included in otherregions of a CAR. In some embodiments, the hinge domain is a CD8 hingedomain. Other hinge domains may be used.

Transmembrane Domain

The transmembrane domain is a hydrophobic alpha helix that spans themembrane. The transmembrane domain provides stability of the CAR. Insome embodiments, the transmembrane domain of a CAR as provided hereinis a CD8 transmembrane domain. In other embodiments, the transmembranedomain is a CD28 transmembrane domain. In yet other embodiments, thetransmembrane domain is a chimera of a CD8 and CD28 transmembranedomain. Other transmembrane domains may be used as provided herein. Insome embodiments, the transmembrane domain is a CD8a transmembranedomain: FVPVFLPAKPTTTPAPRPPTPAPTIAS QPLS LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR (SEQ ID NO: 90). Othertransmembrane domains may be used.

In some embodiments, the transmembrane domain is a CD8a transmembranedomain comprising the amino acid sequence: IYIWAPLAGTCGVLLLSLVITLY (SEQID NO: 126).

Endodomain

The endodomain is the functional end of the receptor. Following antigenrecognition, receptors cluster and a signal is transmitted to the cell.The most commonly used endodomain component is CD3-zeta, which containsthree (3) immunoreceptor tyrosine-based activation motif (ITAM)s. Thistransmits an activation signal to the T cell after the antigen is bound.In many cases, CD3-zeta may not provide a fully competent activationsignal and, thus, a co-stimulatory signaling is used. For example, CD28and/or 4-1BB may be used with CD3-zeta (CD3ζ) to transmit aproliferative/survival signal. Thus, in some embodiments, theco-stimulatory molecule of a CAR as provided herein is a CD28co-stimulatory molecule. In other embodiments, the co-stimulatorymolecule is a 4-1BB co-stimulatory molecule. In some embodiments, a CARincludes CD3ζ and CD28. In other embodiments, a CAR includes CD3-zetaand 4-1BB. In still other embodiments, a CAR includes CD3ζ, CD28, and4-1BB. Table 4 provides examples of signaling domains derived from4-1BB, CD28 and CD3-zeta that may be used herein.

TABLE 4 Name Sequence SEQ ID NO: 4-1BBAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATT  18TATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL  19 CD28TCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACT 121CCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCC SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 20 CD3-zeta CGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCA  21AGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGARVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM  22GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR

Cancer Antigens

CD70

In some embodiments, the T cells of the present disclosure areengineered with a chimeric antigen receptor (CAR) designed to targetCD70. CD70 was initially identified as the ligand for CD27, aco-stimulatory receptor involved in T cell proliferation and survival.CD70 is only found on a small percentage of activated T cells andantigen presenting cells in draining lymph nodes during viral infection.Many human tumors also express CD70 including, but not limited to, solidcancers such as clear cell renal cancer, breast cancer, gastric cancer,ovarian cancer, glioblastoma, and hematological malignancies. Due to itsrestricted expression pattern on normal tissues and overexpression innumerous cancers, CD70 is an attractive therapeutic target.

Thus, in some embodiments, T cells of the present disclosure areengineered to express a CAR comprising an anti-CD70 antibody (e.g.,anti-CD70 scFv). In some embodiments, the anti-CD70 antibody is ananti-CD70 scFv encoded by the sequence of SEQ ID NO: 47 or 49. In someembodiments, the anti-CD70 antibody is an anti-CD70 scFv comprising thesequence of SEQ ID NO: 48 or 50. In some embodiments, the anti-CD70antibody is an anti-CD70 scFv comprising a VH comprising the sequence ofSEQ ID NO: 51. In some embodiments, the anti-CD70 antibody is ananti-CD70 scFv comprising a VL comprising the sequence of SEQ ID NO: 52.In some embodiments, a CAR comprising an anti-CD70 antibody is encodedby the sequence of SEQ ID NO: 45. In some embodiments, a CAR comprisingan anti-CD70 antibody comprises the sequence of SEQ ID NO: 46.

In some embodiments, the anti-CD70 antibody is an anti-CD70 scFv encodedby a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99%identity to SEQ ID NO: 47 or 49. In some embodiments, the anti-CD70antibody is an anti-CD70 scFv comprising an amino acid sequence havingat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97% 98% or 99% identity to SEQ ID NO: 48 or 50.In some embodiments, the anti-CD70 antibody is an anti-CD70 scFvcomprising a VH comprising an amino acid sequence having at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% 98% or 99% identity to SEQ ID NO: 51. In some embodiments,the anti-CD70 antibody is an anti-CD70 scFv comprising a VL comprisingan amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99%identity to SEQ ID NO: 52. In some embodiments, a CAR comprising ananti-CD70 antibody is encoded by a nucleotide sequence having at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98% or 99% identity to SEQ ID NO: 45. In someembodiments, a CAR comprising an anti-CD70 antibody comprises an aminoacid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% identity toSEQ ID NO: 46.

BCMA

In some embodiments, the T cells of the present disclosure areengineered with a CAR designed to target BCMA. B-cell maturation antigen(BCMA, CD269) is a member of the tumor necrosis factor receptor (TNF)superfamily. BCMA binds B-cell activating factor (BAFF) and aproliferation inducing ligand (APRIL). Among nonmalignant cells, BCMA isexpressed primarily by plasma cells and subsets of mature B cells. BCMAis selectively expressed by B-lineage cells including multiple myelomacells and non-Hodgkin's lymphoma, thus BCMA is also an attractivetherapeutic target.

Thus, in some embodiments, T cells of the present disclosure areengineered to express a CAR comprising an anti-BCMA antibody (e.g.,anti-BCMA scFv). In some embodiments, the anti-BCMA antibody is ananti-BCMA scFv encoded by the sequence of SEQ ID NO: 58. In someembodiments, the anti-BCMA antibody is an anti-BCMA scFv comprising thesequence of SEQ ID NO: 59. In some embodiments, the anti-BCMA antibodyis an anti-BCMA scFv comprising a VH comprising the sequence of SEQ IDNO: 60. In some embodiments, the anti-BCMA antibody is an anti-BCMA scFvcomprising a VL comprising the sequence of SEQ ID NO: 61. In someembodiments, a CAR comprising an anti-BCMA antibody is encoded by thesequence of SEQ ID NO: 56. In some embodiments, a CAR comprising ananti-BCMA antibody comprises the sequence of SEQ ID NO: 57.

In some embodiments, the anti-BCMA antibody is an anti-BCMA scFv encodedby a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99%identity to SEQ ID NO: 58. In some embodiments, the anti-BCMA antibodyis an anti-BCMA scFv comprising an amino acid sequence having at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98% or 99% identity to SEQ ID NO: 59. In someembodiments, the anti-BCMA antibody is an anti-BCMA scFv comprising a VHcomprising an amino acid sequence having at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%or 99% identity to SEQ ID NO: 60. In some embodiments, the anti-BCMAantibody is an anti-BCMA scFv comprising a VL comprising an amino acidsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% identity to SEQID NO: 61. In some embodiments, a CAR comprising an anti-BCMA antibodyis encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%or 99% identity to SEQ ID NO: 56. In some embodiments, a CAR comprisingan anti-BCMA antibody comprises an amino acid sequence having at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98% or 99% identity to SEQ ID NO: 57.

CD19

In some embodiments, the T cells of the present disclosure areengineered with a CAR designed to target CD19. Cluster ofDifferentiation 19 (CD19) is an antigenic determinant detectable onleukemia precursor cells. The human and murine amino acid and nucleicacid sequences can be found in a public database, such as GenBank,UniProt and Swiss-Prot. For example, the amino acid sequence of humanCD19 can be found as UniProt/Swiss-Prot Accession No. P15391 and thenucleotide sequence encoding of the human CD19 can be found at AccessionNo. NM—001178098. CD19 is expressed on most B lineage cancers,including, e.g., acute lymphoblastic leukemia, chronic lymphocyteleukemia and non-Hodgkin's lymphoma. It is also an early marker of Bcell progenitors. See, e.g., Nicholson et al. Mol. Immun. 34 (16-17):1157-1165 (1997).

Thus, in some embodiments, T cells of the present disclosure areengineered to express a CAR comprising an anti-CD19 antibody (e.g.,anti-CD19 scFv). In some embodiments, the anti-CD19 antibody is ananti-CD19 scFv encoded by the sequence of SEQ ID NO: 150. In someembodiments, the anti-CD19 antibody is an anti-CD19 scFv comprising thesequence of SEQ ID NO: 151. In some embodiments, the anti-CD19 antibodyis an anti-CD19 scFv comprising a VH comprising the sequence of SEQ IDNO: 152. In some embodiments, the anti-CD19 antibody is an anti-CD19scFv comprising a VL comprising the sequence of SEQ ID NO: 153. In someembodiments, a CAR comprising an anti-CD19 antibody is encoded by thesequence of SEQ ID NO: 148. In some embodiments, a CAR comprising ananti-CD19 antibody comprises the sequence of SEQ ID NO: 149.

In some embodiments, the anti-CD19 antibody is an anti-CD19 scFv encodedby a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99%identity to SEQ ID NO: 150. In some embodiments, the anti-CD19 antibodyis an anti-CD19 scFv comprising an amino acid sequence having at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98% or 99% identity to SEQ ID NO: 151. In someembodiments, the anti-CD19 antibody is an anti-CD19 scFv comprising a VHcomprising an amino acid sequence having at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%or 99% identity to SEQ ID NO: 152. In some embodiments, the anti-CD19antibody is an anti-CD19 scFv comprising a VL comprising an amino acidsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% identity to SEQID NO: 153. In some embodiments, a CAR comprising an anti-CD19 antibodyis encoded by a nucleotide having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99%identity to SEQ ID NO: 148. In some embodiments, a CAR comprising ananti-CD19 antibody comprises an amino acid sequence having at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% 98% or 99% identity to SEQ ID NO: 149.

CD33

In some embodiments, the T cells of the present disclosure areengineered with a CAR designed to target CD33. CD33, also known asSiglec3, is a transmembrane receptor expressed on cells of myeloidlineage that is known to bind sialic acids. As CD33 is expressed incancer cells (e.g., acute myeloid leukemia), it is thought that CD33represents a cell surface marker for targeting these malignancies.

Thus, in some embodiments, T cells of the present disclosure areengineered to express a CAR comprising an anti-CD33 antibody (e.g.,anti-CD33 scFv). In some embodiments, the anti-CD33 antibody is ananti-CD33 scFv encoded by the sequence of SEQ ID NO: 138. In someembodiments, the anti-CD33 antibody is an anti-CD33 scFv comprising thesequence of SEQ ID NO: 137. In some embodiments, the anti-CD33 antibodyis an anti-CD19 scFv comprising a VH comprising the sequence of SEQ IDNO: 140. In some embodiments, the anti-CD33 antibody is an anti-CD33scFv comprising a VL comprising the sequence of SEQ ID NO: 141. In someembodiments, a CAR comprising an anti-CD33 antibody is encoded by thesequence of SEQ ID NO: 136. In some embodiments, a CAR comprising ananti-CD33 antibody comprises the sequence of SEQ ID NO: 139.

In some embodiments, the anti-CD33 antibody is an anti-CD33 scFv encodedby a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99%identity to SEQ ID NO: 138. In some embodiments, the anti-CD33 antibodyis an anti-CD33 scFv comprising an amino acid sequence having at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98% or 99% identity to SEQ ID NO: 137. In someembodiments, the anti-CD33 antibody is an anti-CD19 scFv comprising a VHcomprising an amino acid sequence having at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%or 99% identity to SEQ ID NO: 140. In some embodiments, the anti-CD33antibody is an anti-CD33 scFv comprising a VL comprising an amino acidsequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% identity to SEQID NO: 141. In some embodiments, a CAR comprising an anti-CD33 antibodyis encoded by a nucleotide sequence having at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%or 99% identity to SEQ ID NO: 136. In some embodiments, a CAR comprisingan anti-CD33 antibody comprises an amino acid sequence having at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97% 98% or 99% identity to SEQ ID NO: 139.

Antibodies

An antibody (interchangeably used in plural form) is an immunoglobulinmolecule capable of specific binding to a target, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term “antibody” encompassesnot only intact (i.e., full-length) monoclonal antibodies, but alsoantigen-binding fragments (such as Fab, Fab′, F(ab′)2, Fv), single chainvariable fragment (scFv), mutants thereof, fusion proteins comprising anantibody portion, humanized antibodies, chimeric antibodies, diabodies,linear antibodies, single chain antibodies, single domain antibodies(e.g., camel or llama V_(H)H antibodies), multispecific antibodies(e.g., bispecific antibodies) and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies.

A typical antibody molecule comprises a heavy chain variable region (VH)and a light chain variable region (VL), which are usually involved inantigen binding. These regions/residues that are responsible forantigen-binding can be identified from amino acid sequences of the VH/VLsequences of a reference antibody (e.g., an anti-CD70 antibody or ananti-BCMA antibody as described herein) by methods known in the art. TheVH and VL regions can be further subdivided into regions ofhypervariability, also known as “complementarity determining regions”(“CDR”), interspersed with regions that are more conserved, which areknown as “framework regions” (“FR”). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The extent of the framework region and CDRs can be preciselyidentified using methodology known in the art, for example, by the Kabatdefinition, the Chothia definition, the AbM definition, and/or thecontact definition, all of which are well known in the art. As usedherein, a CDR may refer to the CDR defined by any method known in theart. Two antibodies having the same CDR means that the two antibodieshave the same amino acid sequence of that CDR as determined by the samemethod. See, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, Chothia et al., (1989)Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917,Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J.Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk andbioinf.org.uk/abs.

In some embodiments, an antibody is an scFv, such as an anti-CD70 scFv,an anti-BCMA scFv, an anti-CD19 scFv or an anti-CD33 scFv. An antibodyincludes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM(or sub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantdomain of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The antibodies to be used as provided herein can be murine, rat, human,or any other origin (including chimeric or humanized antibodies). Insome examples, the antibody comprises a modified constant region, suchas a constant region that is immunologically inert, e.g., does nottrigger complement mediated lysis, or does not stimulateantibody-dependent cell mediated cytotoxicity (ADCC).

In some embodiments, an antibody of the present disclosure is ahumanized antibody. Humanized antibodies refer to forms of non-human(e.g., murine) antibodies that are specific chimeric immunoglobulins,immunoglobulin chains, or antigen-binding fragments thereof that containminimal sequence derived from non-human immunoglobulin. For the mostpart, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. A humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,or six) which are altered with respect to the original antibody, whichare also termed one or more CDRs “derived from” one or more CDRs fromthe original antibody. Humanized antibodies may also involve affinitymaturation.

In some embodiments, an antibody of the present disclosure is a chimericantibody, which can include a heavy constant region and a light constantregion from a human antibody.

Chimeric antibodies refer to antibodies having a variable region or partof variable region from a first species and a constant region from asecond species. Typically, in these chimeric antibodies, the variableregion of both light and heavy chains mimics the variable regions ofantibodies derived from one species of mammals (e.g., a non-human mammalsuch as mouse, rabbit, and rat), while the constant portions arehomologous to the sequences in antibodies derived from another mammalsuch as human. In some embodiments, amino acid modifications can be madein the variable region and/or the constant region.

In some embodiments, an antibody of the present disclosure specificallybinds a target antigen, such as human CD70, human BCMA, human CD19 orhuman CD33. An antibody that “specifically binds” to a target or anepitope is a term well understood in the art, and methods to determinesuch specific binding are also well known in the art. A molecule is saidto exhibit “specific binding” if it reacts or associates morefrequently, more rapidly, with greater duration and/or with greateraffinity with a particular target antigen than it does with alternativetargets. An antibody “specifically binds” to a target antigen if itbinds with greater affinity, avidity, more readily, and/or with greaterduration than it binds to other substances. For example, an antibodythat specifically (or preferentially) binds to a CD70, BCMA, CD19 orCD33 epitope is an antibody that binds this CD70, BCMA, CD19 or CD33epitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other CD70, BCMA, CD19 or CD33epitopes or non-CD70, non-BCMA, non-CD19 or non-CD33 epitopes. It isalso understood by reading this definition that, for example, anantibody that specifically binds to a first target antigen may or maynot specifically or preferentially bind to a second target antigen. Assuch, “specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

In some embodiments, the equilibrium dissociation constant (K_(D))between the antibody and CD70 is 100 pM to 1 μM. In some embodiments,the K_(D) between the antibody and CD70 is 1 nM to 100 nM.

In some embodiments, the equilibrium dissociation constant (K_(D))between the antibody and BCMA is 100 pM to 1 μM. In some embodiments,the K_(D) between the antibody and BCMA is 1 nM to 100 nM.

In some embodiments, the equilibrium dissociation constant (K_(D))between the antibody and CD19 is 100 pM to 1 μM. In some embodiments,the K_(D) between the antibody and CD19 is 1 nM to 100 nM.

In some embodiments, the equilibrium dissociation constant (K_(D))between the antibody and CD33 is 100 pM to 1 μM. In some embodiments,the K_(D) between the antibody and CD33 is 1 nM to 100 nM.

Also within the scope of the present disclosure are functional variantsof any of the exemplary antibodies as disclosed herein. A functionalvariant may contain one or more amino acid residue variations in the VHand/or VL, or in one or more of the VH CDRs and/or one or more of the VLCDRs as relative to a reference antibody, while retaining substantiallysimilar binding and biological activities (e.g., substantially similarbinding affinity, binding specificity, inhibitory activity, anti-tumoractivity, or a combination thereof) as the reference antibody.

In some examples, an antibody disclosed herein comprises a VH CDR1, a VHCDR2, and a VH CDR3, which collectively contains no more than 10 aminoacid variations (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) as compared with the VH CDR1, VH CDR2, and VH CDR3 of areference antibody such as Antibody A (VH: SEQ ID NO: 51; VL: SEQ ID NO:52) or Antibody B (VH: SEQ ID NO: 60; VL: SEQ ID NO: 61). “Collectively”means that the total number of amino acid variations in all of the threeVH CDRs is within the defined range. Alternatively or in addition,antibody may comprise a VL CDR1, a VL CDR2, and a VL CDR3, whichcollectively contains no more than 10 amino acid variations (e.g., nomore than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variation) as comparedwith the VL CDR1, VL CDR2, and VL CDR3 of the reference antibody.

In some examples, an antibody disclosed herein may comprise a VH CDR1, aVH CDR2, and a VH CDR3, at least one of which contains no more than 5amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acidvariation) as the counterpart VH CDR of a reference antibody such asAntibody A (VH: SEQ ID NO: 51; VL: SEQ ID NO: 52) or Antibody B (VH: SEQID NO: 60; VL: SEQ ID NO: 61). In specific examples, the antibodycomprises a VH CDR3, which contains no more than 5 amino acid variations(e.g., no more than 4, 3, 2, or 1 amino acid variation) as the VH CDR3of a reference antibody such as Antibody A (VH: SEQ ID NO: 51; VL: SEQID NO: 52) or Antibody B (VH: SEQ ID NO: 60; VL: SEQ ID NO: 61).Alternatively or in addition, an antibody may comprise a VL CDR1, a VLCDR2, and a VL CDR3, at least one of which contains no more than 5 aminoacid variations (e.g., no more than 4, 3, 2, or 1 amino acid variation)as the counterpart VL CDR of the reference antibody. In specificexamples, the antibody comprises a VL CDR3, which contains no more than5 amino acid variations (e.g., no more than 4, 3, 2, or 1 amino acidvariation) as the VL CDR3 of the reference antibody.

In some instances, the amino acid residue variations can be conservativeamino acid residue substitutions. As used herein, a “conservative aminoacid substitution” refers to an amino acid substitution that does notalter the relative charge or size characteristics of the protein inwhich the amino acid substitution is made. Variants can be preparedaccording to methods for altering polypeptide sequence known to one ofordinary skill in the art such as are found in references which compilesuch methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology,F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.Conservative substitutions of amino acids include substitutions madeamongst amino acids within the following groups: (a) A→G, S; (b) R→K, H;(c) N→Q, H; (d) D→E, N; (e) C→S, A; (f) Q→N; (g) E→D, Q; (h) G→A; (i)H→N, Q; (j) I→L, V; (k) L→I, V; (l) K→R, H; (m) M→L, I, Y; (n) F→Y, M,L; (o) P→A; (p) S→T; (q) T→S; (r) W→Y, F; (s) Y→W, F; and (t) V→I, L.

In some embodiments, an antibody disclosed herein may comprise VH CDRsthat collectively are at least 80% (e.g., 85%, 90%, 95%, or 98%)identical to the VH CDRs of a reference antibody such as Antibody A (VH:SEQ ID NO: 51; VL: SEQ ID NO: 52) or Antibody B (VH: SEQ ID NO: 60; VL:SEQ ID NO: 61). Alternatively or in addition, the antibody may compriseVL CDRs that collectively are at least 80% (e.g., 85%, 90%, 95%, or 98%)identical to the VL CDRs of the reference antibody. In some embodiments,an antibody may comprise a VH that is at least 80% (e.g., 85%, 90%, 95%,or 98%) identical to the VH of a reference antibody such as Antibody A(VH: SEQ ID NO: 51; VL: SEQ ID NO: 52) or Antibody B (VH: SEQ ID NO: 60;VL: SEQ ID NO: 61) and/or a VL that is at least 80% (e.g., 85%, 90%,95%, or 98%) identical to the VL of the reference antibody.

In some embodiments, an anti-CD70 antibody (e.g., anti-CD70 scFv)comprises a VH and a VL comprising the amino acid sequences set forth inSEQ ID NOs: 51 and 52, respectively. In some embodiments, an anti-CD70antibody (e.g., anti-CD70 scFv) comprises three CDRs (CDR1, CDR2 andCDR2) of the VH set forth in SEQ ID NO: 51, and three CDRs (CDR1, CDR2and CDR3) of the VL set forth in SEQ ID NO: 52. In some embodiments, ananti-CD70 antibody (e.g., anti-CD70 scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 51, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 52, wherein theCDRs are determined according to Kabat. In some embodiments, ananti-CD70 antibody (e.g., anti-CD70 scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 51, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 52, wherein theCDRs are determined according to Chothia. In some embodiments, ananti-CD70 antibody (e.g., anti-CD70 scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 51, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 52, wherein theCDRs are determined according to AbM. In some embodiments, an anti-CD70antibody (e.g., anti-CD70 scFv) comprises heavy chain CDR1, CDR2 andCDR3 sequences set forth in SEQ ID NOs: 68, 70 and 72, respectively, andlight chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 62,64 and 66. In some embodiments, an anti-CD70 antibody (e.g., anti-CD70scFv) comprises heavy chain CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 69, 71 and 73, respectively, and light chain CDR1, CDR2 andCDR3 sequences set forth in SEQ ID NOs: 63, 65 and 67. In someembodiments, an anti-CD70 antibody is an anti-CD70 scFv comprising theamino acid sequence set forth in SEQ ID NO: 50. In some embodiments, ananti-CD70 antibody is an anti-CD70 scFv encoded by the nucleotidesequence set forth in SEQ ID NO: 49. In some embodiments, an anti-CD70antibody is an anti-CD70 scFv comprising the amino acid sequence setforth in SEQ ID NO: 48. In some embodiments, an anti-CD70 antibody is ananti-CD70 scFv encoded by the nucleotide sequence set forth in SEQ IDNO: 47.

In some embodiments, an anti-BCMA antibody (e.g., anti-BCMA scFv)comprises a VH and a VL comprising the amino acid sequences set forth inSEQ ID NOs: 60 and 61, respectively. In some embodiments, an anti-BCMAantibody (e.g., anti-BCMA scFv) comprises three CDRs (CDR1, CDR2 andCDR2) of the VH set forth in SEQ ID NO: 60, and three CDRs (CDR1, CDR2and CDR3) of the VL set forth in SEQ ID NO: 61. In some embodiments, ananti-BCMA antibody (e.g., anti-BCMA scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 60, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 61, wherein theCDRs are determined according to Kabat. In some embodiments, ananti-BCMA antibody (e.g., anti-BCMA scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 60, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 61, wherein theCDRs are determined according to Chothia. In some embodiments, ananti-BCMA antibody (e.g., anti-BCMA scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 60, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 61, wherein theCDRs are determined according to AbM. In some embodiments, an anti-BCMAantibody (e.g., anti-BCMA scFv) comprises heavy chain CDR1, CDR2 andCDR3 sequences set forth in SEQ ID NOs: 80, 82 and 84, respectively, andlight chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 74,76 and 78. In some embodiments, an anti-BCMA antibody (e.g., anti-BCMAscFv) comprises heavy chain CDR1, CDR2 and CDR3 sequences set forth inSEQ ID NOs: 81, 83 and 85, respectively, and light chain CDR1, CDR2 andCDR3 sequences set forth in SEQ ID NOs: 75, 77 and 79. In someembodiments, an anti-BCMA antibody is an anti-BCMA scFv comprising theamino acid sequence set forth in SEQ ID NO: 59 In some embodiments, ananti-BCMA antibody is an anti-BCMA scFv encoded by the nucleotidesequence set forth in SEQ ID NO: 58.

In some embodiments, an anti-CD19 antibody (e.g., anti-CD19 scFv)comprises a VH and a VL comprising the amino acid sequences set forth inSEQ ID NOs: 152 and 153, respectively. In some embodiments, an anti-CD19antibody (e.g., anti-CD19 scFv) comprises three CDRs (CDR1, CDR2 andCDR2) of the VH set forth in SEQ ID NO: 152, and three CDRs (CDR1, CDR2and CDR3) of the VL set forth in SEQ ID NO: 153. In some embodiments, ananti-CD19 antibody (e.g., anti-CD19 scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 152, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 153, wherein theCDRs are determined according to Kabat. In some embodiments, ananti-CD19 antibody (e.g., anti-CD19 scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 152, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 153, wherein theCDRs are determined according to Chothia. In some embodiments, ananti-CD19 antibody (e.g., anti-CD19 scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 152, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 153, wherein theCDRs are determined according to AbM. In some embodiments, an anti-CD19antibody (e.g., anti-CD19 scFv) comprises heavy chain CDR1, CDR2 andCDR3 sequences set forth in SEQ ID NOs: 169, 170 and 171, respectively,and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:166, 167 and 168, respectively. In some embodiments, an anti-CD19antibody (e.g., anti-CD19 scFv) comprises heavy chain CDR1, CDR2 andCDR3 sequences set forth in SEQ ID NOs: 175, 176 and 177, respectively,and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:172, 173 and 174, respectively. In some embodiments, an anti-CD19antibody is an anti-CD19 scFv comprising the amino acid sequence setforth in SEQ ID NO: 151. In some embodiments, an anti-CD19 antibody isan anti-CD19 scFv encoded by the nucleotide sequence set forth in SEQ IDNO: 150.

In some embodiments, an anti-CD33 antibody (e.g., anti-CD33 scFv)comprises a VH and a VL comprising the amino acid sequences set forth inSEQ ID NOs: 140 and 141, respectively. In some embodiments, an anti-CD33antibody (e.g., anti-CD33 scFv) comprises three CDRs (CDR1, CDR2 andCDR2) of the VH set forth in SEQ ID NO: 140, and three CDRs (CDR1, CDR2and CDR3) of the VL set forth in SEQ ID NO: 141. In some embodiments, ananti-CD33 antibody (e.g., anti-CD33 scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 140, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 141, wherein theCDRs are determined according to Kabat. In some embodiments, ananti-CD33 antibody (e.g., anti-CD33 scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 140, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 141, wherein theCDRs are determined according to Chothia. In some embodiments, ananti-CD33 antibody (e.g., anti-CD33 scFv) comprises three CDRs (CDR1,CDR2 and CDR2) of the VH set forth in SEQ ID NO: 140, and three CDRs(CDR1, CDR2 and CDR3) of the VL set forth in SEQ ID NO: 141, wherein theCDRs are determined according to AbM. In some embodiments, an anti-CD33antibody (e.g., anti-CD33 scFv) comprises heavy chain CDR1, CDR2 andCDR3 sequences set forth in SEQ ID NOs: 142, 143 and 144, respectively,and light chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:145, 146 and 147. In some embodiments, an anti-CD33 antibody (e.g.,anti-CD33 scFv) comprises heavy chain CDR1, CDR2 and CDR3 sequences setforth in SEQ ID NOs: 178, 179 and 180, respectively, and light chainCDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs: 145, 146 and 147.In some embodiments, an anti-CD33 antibody is an anti-CD33 scFvcomprising the amino acid sequence set forth in SEQ ID NO: 137. In someembodiments, an anti-CD33 antibody is an anti-CD33 scFv encoded by thenucleotide sequence set forth in SEQ ID NO: 138.

Antigen Targeting Chimeric Antigen Receptor Construct

In some embodiments, the engineered T cells described herein comprise atumor antigen targeting CAR. In some embodiments, a tumor antigen is a“tumor associated antigen,” referring an immunogenic molecule, such as aprotein, that is generally expressed at a higher level in tumor cellsthan in non-tumor cells, in which it may not be expressed at all, oronly at low levels. In some embodiments, tumor-associated structureswhich are recognized by the immune system of the tumor-harboring hostare referred to as tumor-associated antigens. In some embodiments, atumor-associated antigen is a universal tumor antigen if its broadlyexpressed by most tumors. In some embodiments, tumor-associated antigensare differentiation antigens, mutational antigens, overexpressedcellular antigens or viral antigens. In some embodiments, a tumorantigen is a “tumor specific antigen” or “TSA,” referring to animmunogenic molecule, such as a protein, that is unique to a tumor cell.Tumor specific antigens are exclusively expressed in tumor cells. Insome embodiments, the tumor antigen is not CD70.

In some embodiments, the engineered T cells described herein comprise anon-CD70 targeting CAR (e.g., a CAR that does not bind CD70).

CD19 CAR

In some embodiments, the engineered T cells described herein comprise aCD19 targeting CAR, also referred to herein as CD19 CAR, anti-CD19 CARor anti-CD19 CAR T cells. In some embodiments, the anti-CD19 CARcomprises (i) an ectodomain that comprises an anti-CD19 antigen-bindingdomain, (ii) a transmembrane domain, and (iii) an endodomain comprisingat least one co-stimulatory domain.

In some embodiments, the anti-CD19 CAR comprises (i) an ectodomain thatcomprises an anti-CD19 antigen-binding domain, (ii) a CD8 transmembranedomain, and (iii) an endodomain that comprises a CD28 or 41BBco-stimulatory domain, and a CD3-zeta signaling domain. In someembodiments, the anti-CD19 CAR comprises (i) an ectodomain thatcomprises an anti-CD19 antigen-binding domain, (ii) a CD8 transmembranedomain, and (iii) an endodomain that comprises a CD28 co-stimulatorydomain and a CD3-zeta signaling domain. In some embodiments, theanti-CD19 CAR comprises (i) an ectodomain that comprises an anti-CD19antigen-binding domain, (ii) a CD8 transmembrane domain, and (iii) anendodomain that comprises a 41BB co-stimulatory domain and a CD3-zetasignaling domain.

In some embodiments, the anti-CD19 CAR comprises (i) an ectodomain thatcomprises an anti-CD19 antigen-binding domain, (ii) a CD8 transmembranedomain comprising the amino acid sequence set forth in SEQ ID NO: 126,and (iii) an endodomain that comprises a CD28 co-stimulatory domaincomprising the amino acid sequence set forth in SEQ ID NO: 20 and aCD3-zeta signaling domain comprising the amino acid sequence set forthin SEQ ID NO: 22.

In some embodiments, the anti-CD19 CAR comprises (i) an ectodomain thatcomprises an anti-CD19 scFv comprising the amino acid sequence set forthin SEQ ID NO: 151, (ii) a CD8 transmembrane domain comprising the aminoacid sequence set forth in SEQ ID NO: 126, and (iii) an endodomain thatcomprises a CD28 co-stimulatory domain comprising the amino acidsequence set forth in SEQ ID NO: 20 and a CD3-zeta signaling domaincomprising the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, the anti-CD19 CAR comprises (i) an ectodomain thatcomprises an anti-CD19 scFv comprising variable heavy and light chainregions comprising the amino acid sequences set forth in SEQ ID NOs: 152and 153, respectively, (ii) a CD8 transmembrane domain comprising theamino acid sequence set forth in SEQ ID NO: 126, and (iii) an endodomainthat comprises a CD28 co-stimulatory domain comprising the amino acidsequence set forth in SEQ ID NO: 20 and a CD3-zeta signaling domaincomprising the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, the anti-CD19 CAR comprises the amino acid sequenceset forth in SEQ ID NO: 149. In some embodiments, the anti-CD19 CAR isencoded by the nucleotide sequence set forth in SEQ ID NO: 148. In someembodiments, the anti-CD19 CAR is encoded by a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identityto the nucleotide sequence set forth in SEQ ID NO: 148.

CD33 CAR

In some embodiments, the engineered T cells described herein comprise aCD33 targeting CAR, also referred to herein as CD33 CAR, anti-CD33 CARor anti-CD33 CAR T cells. In some embodiments, the anti-CD33 CARcomprises (i) an ectodomain that comprises an anti-CD33 antigen-bindingdomain, (ii) a transmembrane domain, and (iii) an endodomain comprisingat least one co-stimulatory domain.

In some embodiments, the anti-CD33 CAR comprises (i) an ectodomain thatcomprises an anti-CD33 antigen-binding domain, (ii) a CD8 transmembranedomain, and (iii) an endodomain that comprises a CD28 or 41BBco-stimulatory domain, and a CD3-zeta signaling domain. In someembodiments, the anti-CD33 CAR comprises (i) an ectodomain thatcomprises an anti-CD33 antigen-binding domain, (ii) a CD8 transmembranedomain, and (iii) an endodomain that comprises a CD28 co-stimulatorydomain and a CD3-zeta signaling domain. In some embodiments, theanti-CD33 CAR comprises (i) an ectodomain that comprises an anti-CD33antigen-binding domain, (ii) a CD8 transmembrane domain, and (iii) anendodomain that comprises a 41BB co-stimulatory domain and a CD3-zetasignaling domain.

In some embodiments, the anti-CD33 CAR comprises (i) an ectodomain thatcomprises an anti-CD33 antigen-binding domain, (ii) a CD8 transmembranedomain comprising the amino acid sequence set forth in SEQ ID NO: 126,and (iii) an endodomain that comprises a 41BB co-stimulatory domaincomprising the amino acid sequence set forth in SEQ ID NO: 19 and aCD3-zeta signaling domain comprising the amino acid sequence set forthin SEQ ID NO: 22.

In some embodiments, the anti-CD33 CAR comprises (i) an ectodomain thatcomprises an anti-CD33 scFv comprising the amino acid sequence set forthin SEQ ID NO: 137, (ii) a CD8 transmembrane domain comprising the aminoacid sequence set forth in SEQ ID NO: 126, and (iii) an endodomain thatcomprises a 41BB co-stimulatory domain comprising the amino acidsequence set forth in SEQ ID NO: 19 and a CD3-zeta signaling domaincomprising the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, the anti-CD33 CAR comprises (i) an ectodomain thatcomprises an anti-CD33 scFv comprising variable heavy and light chainregions comprising the amino acid sequences set forth in SEQ ID NOs: 140and 141, respectively, (ii) a CD8 transmembrane domain comprising theamino acid sequence set forth in SEQ ID NO: 126, and (iii) an endodomainthat comprises a 41BB co-stimulatory domain comprising the amino acidsequence set forth in SEQ ID NO: 19 and a CD3-zeta signaling domaincomprising the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, the anti-CD33 CAR comprises the amino acid sequenceset forth in SEQ ID NO: 139. In some embodiments, the anti-CD33 CAR isencoded by the nucleotide sequence set forth in SEQ ID NO: 136. In someembodiments, the anti-CD33 CAR is encoded by a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identityto the nucleotide sequence set forth in SEQ ID NO: 136.

BCMA CAR

In some embodiments, the engineered T cells described herein comprise aBCMA targeting CAR, also referred to herein as BCMA CAR, anti-BCMA CARor anti-BCMA CAR T cells. In some embodiments, the anti-BCMA CARcomprises (i) an ectodomain that comprises an anti-BCMA antigen-bindingdomain, (ii) a transmembrane domain, and (iii) an endodomain comprisingat least one co-stimulatory domain.

In some embodiments, the anti-BCMA CAR comprises (i) an ectodomain thatcomprises an anti-BCMA antigen-binding domain, (ii) a CD8 transmembranedomain, and (iii) an endodomain that comprises a CD28 or 41BBco-stimulatory domain, and a CD3-zeta signaling domain. In someembodiments, the anti-BCMA CAR comprises (i) an ectodomain thatcomprises an anti-BCMA antigen-binding domain, (ii) a CD8 transmembranedomain, and (iii) an endodomain that comprises a CD28 co-stimulatorydomain and a CD3-zeta signaling domain. In some embodiments, theanti-BCMA CAR comprises (i) an ectodomain that comprises an anti-BCMAantigen-binding domain, (ii) a CD8 transmembrane domain, and (iii) anendodomain that comprises a 41BB co-stimulatory domain and a CD3-zetasignaling domain.

In some embodiments, the anti-BCMA CAR comprises (i) an ectodomain thatcomprises an anti-BCMA antigen-binding domain, (ii) a CD8 transmembranedomain comprising the amino acid sequence set forth in SEQ ID NO: 126,and (iii) an endodomain that comprises a 41BB co-stimulatory domaincomprising the amino acid sequence set forth in SEQ ID NO: 19 and aCD3-zeta signaling domain comprising the amino acid sequence set forthin SEQ ID NO: 22.

In some embodiments, the anti-BCMA CAR comprises (i) an ectodomain thatcomprises an anti-BCMA scFv comprising the amino acid sequence set forthin SEQ ID NO: 59, (ii) a CD8 transmembrane domain comprising the aminoacid sequence set forth in SEQ ID NO: 126, and (iii) an endodomain thatcomprises a 41BB co-stimulatory domain comprising the amino acidsequence set forth in SEQ ID NO: 19 and a CD3-zeta signaling domaincomprising the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, the anti-BCMA CAR comprises (i) an ectodomain thatcomprises an anti-BCMA scFv comprising variable heavy and light chainregions comprising the amino acid sequences set forth in SEQ ID NOs: 60and 61, respectively, (ii) a CD8 transmembrane domain comprising theamino acid sequence set forth in SEQ ID NO: 126, and (iii) an endodomainthat comprises a 41BB co-stimulatory domain comprising the amino acidsequence set forth in SEQ ID NO: 19 and a CD3-zeta signaling domaincomprising the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, the anti-BCMA CAR comprises the amino acid sequenceset forth in SEQ ID NO: 57. In some embodiments, the anti-BCMA CAR isencoded by the nucleotide sequence set forth in SEQ ID NO: 56. In someembodiments, the anti-BCMA CAR is encoded by a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identityto the nucleotide sequence set forth in SEQ ID NO: 56.

CD70 CAR

In some embodiments, the engineered T cells described herein comprise aCD70 targeting CAR, also referred to herein as CD70 CAR, anti-CD70 CARor anti-CD70 CAR T cells. In some embodiments, the anti-CD70 CARcomprises (i) an ectodomain that comprises an anti-CD70 antigen-bindingdomain, (ii) a transmembrane domain, and (iii) an endodomain comprisingat least one co-stimulatory domain.

In some embodiments, the anti-CD70 CAR comprises (i) an ectodomain thatcomprises an anti-CD70 antigen-binding domain, (ii) a CD8 transmembranedomain, and (iii) an endodomain that comprises a CD28 or 41BBco-stimulatory domain, and a CD3-zeta signaling domain. In someembodiments, the anti-CD70 CAR comprises (i) an ectodomain thatcomprises an anti-CD70 antigen-binding domain, (ii) a CD8 transmembranedomain, and (iii) an endodomain that comprises a CD28 co-stimulatorydomain and a CD3-zeta signaling domain. In some embodiments, theanti-CD70 CAR comprises (i) an ectodomain that comprises an anti-CD70antigen-binding domain, (ii) a CD8 transmembrane domain, and (iii) anendodomain that comprises a 41BB co-stimulatory domain and a CD3-zetasignaling domain.

In some embodiments, the anti-CD70 CAR comprises (i) an ectodomain thatcomprises an anti-CD70 antigen-binding domain, (ii) a CD8 transmembranedomain comprising the amino acid sequence set forth in SEQ ID NO: 126,and (iii) an endodomain that comprises a 41BB co-stimulatory domaincomprising the amino acid sequence set forth in SEQ ID NO: 19 and aCD3-zeta signaling domain comprising the amino acid sequence set forthin SEQ ID NO: 22.

In some embodiments, the anti-CD70 CAR comprises (i) an ectodomain thatcomprises an anti-CD70 scFv comprising the amino acid sequence set forthin SEQ ID NO: 50, (ii) a CD8 transmembrane domain comprising the aminoacid sequence set forth in SEQ ID NO: 126, and (iii) an endodomain thatcomprises a 41BB co-stimulatory domain comprising the amino acidsequence set forth in SEQ ID NO: 19 and a CD3-zeta signaling domaincomprising the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, the anti-CD70 CAR comprises (i) an ectodomain thatcomprises an anti-CD70 scFv comprising variable heavy and light chainregions comprising the amino acid sequences set forth in SEQ ID NOs: 51and 52, respectively, (ii) a CD8 transmembrane domain comprising theamino acid sequence set forth in SEQ ID NO: 126, and (iii) an endodomainthat comprises a 41BB co-stimulatory domain comprising the amino acidsequence set forth in SEQ ID NO: 19 and a CD3-zeta signaling domaincomprising the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, the anti-CD70 CAR comprises the amino acid sequenceset forth in SEQ ID NO: 46. In some embodiments, the anti-CD70 CAR isencoded by the nucleotide sequence set forth in SEQ ID NO: 45. In someembodiments, the anti-CD70 CAR is encoded by a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identityto the nucleotide sequence set forth in SEQ ID NO: 45.

Expression of Chimeric Antigen Receptor Construct

Donor Template

The nucleic acid encoding a CAR may be delivered to a T cell thatcomprises what is referred to herein as a donor template (also referredto as a donor polynucleotide). A donor template can contain anon-homologous sequence, such as the nucleic acid encoding a CAR,flanked by two regions of homology to allow for efficient HDR at agenomic location of interest. Alternatively, a donor template may haveno regions of homology to the targeted location in the DNA and may beintegrated by NHEJ-dependent end joining following cleavage at thetarget site.

A donor template can be DNA or RNA, single-stranded and/ordouble-stranded, and can be introduced into a cell in linear or circularform. If introduced in linear form, the ends of the donor sequence canbe protected (e.g., from exonucleolytic degradation) by methods known tothose of skill in the art. For example, one or more dideoxynucleotideresidues are added to the 3′ terminus of a linear molecule and/orself-complementary oligonucleotides are ligated to one or both ends.See, for example, Chang et al., (1987) Proc. Natl. Acad. Sci. USA84:4959-4963; Nehls et al., (1996) Science 272:886-889. Additionalmethods for protecting exogenous polynucleotides from degradationinclude, but are not limited to, addition of terminal amino group(s) andthe use of modified internucleotide linkages such as, for example,phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyriboseresidues.

A donor template can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover, adonor template can be introduced as naked nucleic acid, as nucleic acidcomplexed with an agent such as a liposome or poloxamer, or can bedelivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus,lentivirus and integrase defective lentivirus (IDLV)).

A donor template, in some embodiments, is inserted so that itsexpression is driven by the endogenous promoter at the integration site,namely the promoter that drives expression of the endogenous gene intowhich the donor is inserted. However, in some embodiments, the donortemplate comprises an exogenous promoter and/or enhancer, for example aconstitutive promoter, an inducible promoter, or tissue-specificpromoter. In some embodiments, the exogenous promoter is an EF1αpromoter comprising a sequence of SEQ ID NO: 123. Other promoters may beused.

Furthermore, exogenous sequences may also include transcriptional ortranslational regulatory sequences, for example, promoters, enhancers,insulators, internal ribosome entry sites, sequences encoding 2Apeptides and/or polyadenylation signals.

In some embodiments, the donor template comprises a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 98% identity to SEQID NO: 44. In some embodiments, the donor template comprises thenucleotide sequence of SEQ ID NO: 44.

In some embodiments, the donor template comprises a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 98% identity to SEQID NO: 55. In some embodiments, the donor template comprises thenucleotide sequence of SEQ ID NO: 55.

In some embodiments, the donor template comprises a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 98% identity to SEQID NO: 135. In some embodiments, the donor template comprises thenucleotide sequence of SEQ ID NO: 135.

In some embodiments, the donor template comprises a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 98% identity to SEQID NO: 156. In some embodiments, the donor template comprises thenucleotide sequence of SEQ ID NO: 156.

Other Methods

In some embodiments, a nucleic acid encoding a CAR is introduced into anengineered cell by methods known to those of skill in the art. Forexample, a CAR may be introduced into an engineered cell by a vector. Avariety of different methods known in the art can be used to introduceany of the nucleic acids or expression vectors disclosed herein into animmune effector cell. Non-limiting examples of methods for introducingnucleic acid into a cell include: lipofection, transfection (e.g.,calcium phosphate transfection, transfection using highly branchedorganic compounds, transfection using cationic polymers, dendrimer-basedtransfection, optical transfection, particle-based transfection (e.g.,nanoparticle transfection), or transfection using liposomes (e.g.,cationic liposomes)), microinjection, electroporation, cell squeezing,sonoporation, protoplast fusion, impalefection, hydrodynamic delivery,gene gun, magnetofection, viral transfection, and nucleofection.

Delivery Methods and Constructs

Nucleases and/or donor templates may be delivered using a vector system,including, but not limited to, plasmid vectors, DNA minicircles,retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirusvectors; herpesvirus vectors and adeno-associated virus vectors, andcombinations thereof.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor templates incells (e.g., T cells). Non-viral vector delivery systems include DNAplasmids, DNA minicircles, naked nucleic acid, and nucleic acidcomplexed with a delivery vehicle such as a liposome or poloxamer. Viralvector delivery systems include DNA and RNA viruses, which have eitherepisomal or integrated genomes after delivery to the cell.

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,naked RNA, capped RNA, artificial virions, and agent-enhanced uptake ofDNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) canalso be used for delivery of nucleic acids. Some specific examples areprovided below.

Adeno-Associated Viral Delivery

The donor nucleic acid encoding a CAR construct can be delivered to acell using an adeno-associated virus (AAV). AAVs are small viruses whichintegrate site-specifically into the host genome and can thereforedeliver a transgene, such as CAR. Inverted terminal repeats (ITRs) arepresent flanking the AAV genome and/or the transgene of interest andserve as origins of replication. Also present in the AAV genome are repand cap proteins which, when transcribed, form capsids which encapsulatethe AAV genome for delivery into target cells. Surface receptors onthese capsids which confer AAV serotype, which determines which targetorgans the capsids will primarily bind and thus what cells the AAV willmost efficiently infect. There are twelve currently known human AAVserotypes. In some embodiments, the AAV is AAV serotype 6 (AAV6).

Adeno-associated viruses are among the most frequently used viruses forgene therapy for several reasons. First, AAVs do not provoke an immuneresponse upon administration to mammals, including humans. Second, AAVsare effectively delivered to target cells, particularly whenconsideration is given to selecting the appropriate AAV serotype.Finally, AAVs have the ability to infect both dividing and non-dividingcells because the genome can persist in the host cell withoutintegration. This trait makes them an ideal candidate for gene therapy.

Homology-Directed Repair (HDR)

The donor nucleic acid encoding a CAR is inserted by homology directedrepair (HDR) into the target gene locus. Both strands of the DNA at thetarget locus are cut by a CRISPR Cas9 enzyme. HDR then occurs to repairthe double-strand break (DSB) and insert the donor DNA. For this tooccur correctly, the donor sequence is designed with flanking residueswhich are complementary to the sequence surrounding the DSB site in thetarget gene (hereinafter “homology arms”). These homology arms serve asthe template for DSB repair and allow HDR to be an essentiallyerror-free mechanism. The rate of homology directed repair (HDR) is afunction of the distance between the mutation and the cut site sochoosing overlapping or nearby target sites is important. Templates caninclude extra sequences flanked by the homologous regions or can containa sequence that differs from the genomic sequence, thus allowingsequence editing.

The target gene can be associated with an immune response in a subject,wherein permanently deleting at least a portion of the target gene willmodulate the immune response. For example, to generate a CAR T cell, thetarget gene can be the TCRα constant region (TRAC). Disruption of TRACleads to loss of function of the endogenous TCR.

In some embodiments, the target gene is in a safe harbor locus.

Engineered T Cells

Engineered (gene edited) CAR T cells of the present disclosure may beautologous (“self”) or non-autologous (“non-self,” e.g., allogeneic,syngeneic or xenogeneic). “Autologous” refers to cells from the samesubject. “Allogeneic” refers to cells of the same species as a subject,but that differ genetically to the cells in the subject. In someembodiments, the T cells are obtained from a mammal. In someembodiments, the T cells are obtained from a human.

T cells can be obtained from a number of sources including, but notlimited to, peripheral blood mononuclear cells, bone marrow, lymph nodestissue, cord blood, thymus issue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. In certainembodiments, T cells can be obtained from a unit of blood collected froma subject using any number of techniques known to the skilled person,such as sedimentation, e.g., FICOLL™ separation.

In some embodiments, an isolated population of T cells is used. In someembodiments, after isolation of peripheral blood mononuclear cells(PBMC), both cytotoxic and helper T lymphocytes can be sorted intonaive, memory, and effector T cell subpopulations either before or afteractivation, expansion, and/or genetic modification.

A specific subpopulation of T cells, expressing one or more of thefollowing cell surface markers: TCRab, CD3, CD4, CD8, CD27 CD28, CD38CD45RA, CD45RO, CD62L, CD127, CD122, CD95, CD197, CCR7, KLRG1, MCH-Iproteins and/or MCH-II proteins, can be further isolated by positive ornegative selection techniques. In some embodiments, a specificsubpopulation of T cells, expressing one or more of the markers selectedfrom the group consisting of TCRab, CD4 and/or CD8, is further isolatedby positive or negative selection techniques. In some embodiments, theengineered T cell populations do not express or do not substantiallyexpress one or more of the following markers: CD70, CD57, CD244, CD160,PD-1, CTLA4, HM3, and LAG3. In some embodiments, subpopulations of Tcells may be isolated by positive or negative selection prior to geneticengineering and/or post genetic engineering.

In some embodiments, an isolated population of T cells expresses one ormore of the markers including, but not limited to a CD3+, CD4+, CD8+, ora combination thereof. In some embodiments, the T cells are isolatedfrom a donor, or subject, and first activated and stimulated toproliferate in vitro prior to undergoing gene editing.

To achieve sufficient therapeutic doses of T cell compositions, T cellsare often subjected to one or more rounds of stimulation, activationand/or expansion. T cells can be activated and expanded generally usingmethods as described, for example, in U.S. Pat. Nos. 6,352,694;6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681;7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223;6,905,874; 6,797,514; and 6,867,041. In some embodiments, T cells areactivated and expanded for about 1 day to about 4 days, about 1 day toabout 3 days, about 1 day to about 2 days, about 2 days to about 3 days,about 2 days to about 4 days, about 3 days to about 4 days, or about 1day, about 2 days, about 3 days, or about 4 days prior to introductionof the genome editing compositions into the T cells.

In some embodiments, T cells are activated and expanded for about 4hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours,about 36 hours, about 48 hours, about 60 hours, or about 72 hours priorto introduction of the gene editing compositions into the T cells.

In some embodiments, T cells are activated at the same time that genomeediting compositions are introduced into the T cells. T cell populationsor isolated T cells generated by any of the gene editing methodsdescribed herein are also within the scope of the present disclosure.

In some embodiments, provided herein is a population of T cellscomprising genetically engineered T cells, which comprise a disruptedendogenous CD70 gene and a nucleic acid encoding a chimeric antigenreceptor (CAR), e.g., those described herein. In some embodiments, theCAR binds an antigen expressed on a pathological cell. In someembodiments, the CAR binds CD70. In other embodiments, the CAR does notbind CD70. Such a T cell population may further comprise geneticallyengineered T cells having one or more of the following gene edits: adisrupted endogenous programmed cell death-1 (PD-1) gene, a disruptedendogenous T cell receptor alpha chain constant region (TRAC) gene, anda disrupted endogenous beta-2-microglobulin (β2M) gene. In someexamples, the nucleic acid encoding the CAR may be inserted into theTRAC locus. In some embodiments, the population of T cells disclosedherein comprises genetically engineered T cells, which comprise adisrupted CD70 gene and a nucleic acid encoding a chimeric antigenreceptor (CAR) that binds an antigen expressed on a pathological cell.In some embodiments, the population of T cells disclosed hereincomprises genetically engineered T cells, which comprise a disruptedCD70 gene and a nucleic acid encoding a chimeric antigen receptor (CAR),wherein the CAR binds CD70. In other embodiments, the population of Tcells disclosed herein comprises genetically engineered T cells thatcomprise a disrupted CD70 gene and a nucleic acid encoding a CAR,wherein the CAR does not bind CD70. In some embodiments, the populationof T cells disclosed herein comprises genetically engineered T cells,which comprise a disrupted CD70 gene and a nucleic acid encoding achimeric antigen receptor (CAR) that binds an antigen expressed on apathological cell, and further comprises a disrupted PD1 gene. In someembodiments, the CAR binds CD70. In some embodiments the CAR does notbind CD70. In some aspects, the CAR binds CD19. In some embodiments, theCAR binds CD33. In some aspects, the CAR binds BCMA. Any of thejust-noted engineered T cells may further comprise a disrupted T cellreceptor alpha chain constant region (TRAC) gene and/or a disruptedbeta-2-microglobulin (β2M) gene.

In particular examples, provided herein is a population of T cellscomprising genetically engineered T cells, which comprise a disruptedCD70 gene, a disrupted T cell receptor alpha chain constant region(TRAC) gene, a disrupted beta-2-microglobulin (β2M) gene, a nucleic acidencoding a chimeric antigen receptor (CAR), e.g., an anti-BCMA CAR,anti-CD19 CAR, anti-CD33 CAR, or anti-CD70 CAR as described herein, andoptionally a disrupted programmed cell death-1 (PD-1) gene. Any of theengineered T cells disclosed herein may contain native (undisrupted) HLAgenes.

In some examples, at least 50% (e.g., 60%, 70%, 80%, 90%, or 95%) of thepopulation of T cells express the CAR as disclosed herein and do notexpress a detectable level of surface CD70. Such cells may furtherpossess the features of not expressing a detectable level of surfaceTCR, a detectable level of surface β2M, and/or a detectable level ofsurface PD-1. For example, at least 50% (e.g., 60%, 70%, 80%, 90%, or95%) of the population of T cells express the CAR as disclosed hereinand do not express a detectable level of surface CD70, a detectablelevel of surface TCR, and a detectable level of surface β2M. In someinstances, at least 50% (e.g., 60%, 70%, 80%, 90%, or 95%) of thepopulation of T cells express the CAR as disclosed herein and do notexpress a detectable level of surface CD70, a detectable level ofsurface TCR, a detectable level of surface β2M, and a detectable levelof PD-1.

An isolated cell expressing the CAR as described herein and does notexpress a detectable level of surface CD70 is also within the scope ofthe present disclosure. Such an isolated cell may not express adetectable level of surface TCR, a detectable level of surface β2M,and/or a detectable level of surface PD-1. In some examples, theisolated cell comprises a nucleic acid encoding the CAR, which isinserted into the TRAC locus.

Also provided herein are an engineered T cell population comprisingengineered T cells comprising an RNA-guided nuclease, e.g., thosedescribed herein (for example, a Cas9 nuclease), and a guide RNA (gRNA)targeting a CD70 gene (e.g., those described herein). In some instances,at least 50% (e.g., 60%, 70%, 80%, 90%, or 95%) of the T cells in the Tcell population comprise the RNA-guided nuclease and the gRNA targetingthe CD70 gene. Such an engineered T cell population may further compriseengineered T cells comprising a gRNA targeting a PD-1 gene, a gRNAtargeting a TRAC gene, a gRNA targeting a β2M gene, and/or a nucleicacid (e.g., a vector) comprising a donor template that comprises anucleotide sequence encoding a CAR (e.g., those described herein), whichoptionally is flanked by left and right homology arms to the TRAC genelocus. In some examples, at least 50% (e.g., 60%, 70%, 80%, 90%, or 95%)of the T cells in the T cell population comprise the RNA-guidednuclease, the gRNA targeting the CD70 gene, and the nucleic acid codingfor the CAR. When the nucleic acid coding for the CAR further comprisesthe left and right homology arms to the TRAC gene locus, the T cells mayalso comprise a gRNA targeting the TRAC gene. In addition, the T cellsmay further comprise a gRNA targeting a PD-1 gene, a gRNA targeting aβ2M gene, or a combination thereof.

Also within the scope of the present disclosure is an isolatedengineered T cell comprising the RNA-guided nuclease, the gRNA targetingthe CD70 gene, and optionally one or more of a gRNA targeting a PD-1gene, a gRNA targeting a TRAC gene, a gRNA targeting a β2M gene, and anucleic acid (e.g., a vector) comprising a donor template that comprisesa nucleotide sequence encoding a CAR (e.g., those described herein). Thenucleotide sequence encoding the CAR may be flanked by left and righthomology arms to the TRAC gene locus.

Generating CAR-T Cells

In some embodiments, the engineered T cells described herein aregenerated by modifying the genome of the cells. In some embodiments, adouble stranded break (DSB) at a site in a target gene is induced. Insome embodiments, the DSB is repaired using one or more endogenous DNArepair pathways. In some embodiments, a DNA repair pathway does notrequire a homologous sequence (e.g., the non-homologous end joiningpathway or NHEJ pathway). In some embodiments, a repair pathway requiresa homologous sequence (e.g., the homology-directed pathway or HDRpathway).

In some embodiments, the engineered T cells described herein aregenerated by inducing a DSB with CRISPR-Cas9 as an endonuclease, and oneor more non-coding RNAs, and repairing the DSB using HDR and a donorpolynucleotide template described herein.

In some embodiments, the engineered T cells described herein aregenerated using a gRNA complimentary to a sequence of a target gene thatis a TRAC. In some embodiments, the engineered T cells described hereinare generated using a TRAC gRNA spacer comprising the sequence set forthin SEQ ID NO: 98. In some embodiments, the engineered T cells describedherein are generated using a TRAC gRNA comprising the sequence set forthin SEQ ID NO: 30. In some embodiments, the TRAC gRNA comprising thesequence set forth in SEQ ID NO: 98 targets the TRAC sequence set forthin SEQ ID NO: 118. In some embodiments, the TRAC gRNA comprising thesequence set forth in SEQ ID NO: 30 targets the TRAC sequence set forthin SEQ ID NO: 118.

In some embodiments, the engineered T cells described herein aregenerated using a TRAC gRNA spacer comprising the sequence set forth inSEQ ID NO: 108. In some embodiments, the engineered T cells describedherein are generated using a TRAC gRNA comprising the sequence set forthin SEQ ID NO: 40. In some embodiments, the TRAC gRNA comprising thesequence set forth in SEQ ID NO: 108 targets the TRAC sequence set forthin SEQ ID NO: 118. In some embodiments, the TRAC gRNA comprising thesequence set forth in SEQ ID NO: 40 targets the TRAC sequence set forthin SEQ ID NO: 118.

In some embodiments, the engineered T cells described herein aregenerated using a gRNA complimentary to a sequence of a target gene thatis a β2M. In some embodiments, the engineered T cells described hereinare generated using a β2M gRNA spacer comprising the sequence set forthin SEQ ID NO: 99. In some embodiments, the engineered T cells describedherein are generated using a β2M gRNA comprising the sequence set forthin SEQ ID NO: 31. In some embodiments, the β2M gRNA comprising thesequence set forth in SEQ ID NO: 99 targets the β2M sequence set forthin SEQ ID NO: 119. In some embodiments, the β2M gRNA comprising thesequence set forth in SEQ ID NO: 31 targets the β2M sequence set forthin SEQ ID NO: 119.

In some embodiments, the engineered T cells described herein aregenerated using a β2M gRNA spacer comprising the sequence set forth inSEQ ID NO: 109. In some embodiments, the engineered T cells describedherein are generated using a β2M gRNA comprising the sequence set forthin SEQ ID NO: 41. In some embodiments, the β2M gRNA comprising thesequence set forth in SEQ ID NO: 109 targets the β2M sequence set forthin SEQ ID NO: 119. In some embodiments, the β2M gRNA comprising thesequence set forth in SEQ ID NO: 41 targets the β2M sequence set forthin SEQ ID NO: 119.

In some embodiments, the engineered T cells described herein aregenerated using a gRNA complimentary to a sequence of a target gene thatis a CD70. In some embodiments, the engineered T cells described hereinare generated using a CD70 gRNA spacer comprising the sequence set forthin SEQ ID NO: 94. In some embodiments, the engineered T cells describedherein are generated using a CD70 gRNA comprising the sequence set forthin SEQ ID NO: 26. In some embodiments, the CD70 gRNA comprising thesequence set forth in SEQ ID NO: 94 targets the CD70 sequence set forthin SEQ ID NO: 114. In some embodiments, the CD70 gRNA comprising thesequence set forth in SEQ ID NO: 26 targets the CD70 sequence set forthin SEQ ID NO: 114.

In some embodiments, the engineered T cells described herein aregenerated using a CD70 gRNA spacer comprising the sequence set forth inSEQ ID NO: 104. In some embodiments, the engineered T cells describedherein are generated using a CD70 gRNA comprising the sequence set forthin SEQ ID NO: 36. In some embodiments, the CD70 gRNA comprising thesequence set forth in SEQ ID NO: 104 targets the CD70 sequence set forthin SEQ ID NO: 114. In some embodiments, the CD70 gRNA comprising thesequence set forth in SEQ ID NO: 36 targets the CD70 sequence set forthin SEQ ID NO: 114.

In some embodiments, the engineered T cells described herein aregenerated using a gRNA complimentary to a sequence of a target gene thatis a CD70. In some embodiments, the engineered T cells described hereinare generated using a CD70 gRNA spacer comprising the sequence set forthin SEQ ID NO: 95. In some embodiments, the engineered T cells describedherein are generated using a CD70 gRNA comprising the sequence set forthin SEQ ID NO: 27. In some embodiments, the CD70 gRNA comprising thesequence set forth in SEQ ID NO: 95 targets the CD70 sequence set forthin SEQ ID NO: 115. In some embodiments, the CD70 gRNA comprising thesequence set forth in SEQ ID NO: 27 targets the CD70 sequence set forthin SEQ ID NO: 115.

In some embodiments, the engineered T cells described herein aregenerated using a CD70 gRNA spacer comprising the sequence set forth inSEQ ID NO: 105. In some embodiments, the engineered T cells describedherein are generated using a CD70 gRNA comprising the sequence set forthin SEQ ID NO: 37. In some embodiments, the CD70 gRNA comprising thesequence set forth in SEQ ID NO: 105 targets the CD70 sequence set forthin SEQ ID NO: 115. In some embodiments, the CD70 gRNA comprising thesequence set forth in SEQ ID NO: 37 targets the CD70 sequence set forthin SEQ ID NO: 115.

In some embodiments, the engineered T cells described herein aregenerated using a gRNA complimentary to a sequence of a target gene thatis a PD-1. In some embodiments, the engineered T cells described hereinare generated using a PD-1 gRNA spacer comprising the sequence set forthin SEQ ID NO: 100. In some embodiments, the engineered T cells describedherein are generated using a PD-1 gRNA comprising the sequence set forthin SEQ ID NO: 32. In some embodiments, the PD-1 gRNA comprising thesequence set forth in SEQ ID NO: 100 targets the β2M sequence set forthin SEQ ID NO: 120. In some embodiments, the PD-1 gRNA comprising thesequence set forth in SEQ ID NO: 32 targets the PD-1 sequence set forthin SEQ ID NO: 120.

In some embodiments, the engineered T cells described herein aregenerated using a PD-1 gRNA spacer comprising the sequence set forth inSEQ ID NO: 110. In some embodiments, the engineered T cells describedherein are generated using a PD-1 gRNA comprising the sequence set forthin SEQ ID NO: 42. In some embodiments, the PD-1 gRNA comprising thesequence set forth in SEQ ID NO: 110 targets the PD-1 sequence set forthin SEQ ID NO: 120. In some embodiments, the PD-1 gRNA comprising thesequence set forth in SEQ ID NO: 42 targets the PD-1 sequence set forthin SEQ ID NO: 120.

In some embodiments, the engineered T cells described herein aregenerated using a TRAC gRNA comprising the sequence set forth in SEQ IDNO: 98, a β2M gRNA comprising the sequence set forth in SEQ ID NO: 99, aCD70 gRNA comprising the sequence set forth in SEQ ID NO: 94 or 95,and/or a PD-1 gRNA comprising the sequence set forth in SEQ ID NO: 100.In some embodiments, the engineered T cells described herein aregenerated using a TRAC gRNA comprising the sequence set forth in SEQ IDNO: 108, a β2M gRNA comprising the sequence set forth in SEQ ID NO: 109,a CD70 gRNA comprising the sequence set forth in SEQ ID NO: 104 or 105,and/or a PD-1 gRNA comprising the sequence set forth in SEQ ID NO: 110.

In some embodiments, the engineered T cells described herein aregenerated using a TRAC gRNA comprising the sequence set forth in SEQ IDNO: 30, a β2M gRNA comprising the sequence set forth in SEQ ID NO: 31, aCD70 gRNA comprising the sequence set forth in SEQ ID NO: 26 or 27,and/or a PD-1 gRNA comprising the sequence set forth in SEQ ID NO: 32.In some embodiments, the engineered T cells described herein aregenerated using a TRAC gRNA comprising the sequence set forth in SEQ IDNO: 40, a β2M gRNA comprising the sequence set forth in SEQ ID NO: 41, aCD70 gRNA comprising the sequence set forth in SEQ ID NO: 36 or 27,and/or a PD-1 gRNA comprising the sequence set forth in SEQ ID NO: 42.

In some embodiments, the engineered T cells are generated using a donortemplate comprising a non-homologous sequence that is a nucleic acidencoding a CAR. In some embodiments, a donor template is comprised ofhomology arms that correspond to sequences in a target gene that is aTRAC. In some embodiments, a 5′ homology arm (left homology arm) of thedonor template comprises the sequence set forth in SEQ ID NO: 122. Insome embodiments, a 3′ homology arm of the donor template comprises thesequence set forth in SEQ ID NO: 125.

In some embodiments, an exogenous promoter is an EF1α promoter comprisesthe sequence set forth in SEQ ID NO: 123. In some embodiments, a donortemplate comprises the sequence set forth in SEQ ID NO: 135. In someembodiments, a donor template comprises the sequence set forth in SEQ IDNO: 156. In some embodiments, a donor template comprises the sequenceset forth in SEQ ID NO: 44. In some embodiments, a donor templatecomprises the sequence set forth in SEQ ID NO: 55.

In some embodiments, polynucleotides encoding gRNAs, nucleases, anddonor templates are introduced into cells (e.g., T cells) usingconventional viral and non-viral based gene transfer methods.

In some embodiments, a polynucleotide such as a gRNA, a sgRNA, an mRNAencoding a nuclease, or a donor template are delivered to a cell using anon-viral vector delivery system. Examples of a non-viral vectordelivery system include, but are not limited to, a DNA plasmid, a DNAminicircle, a naked nucleic acid, a liposome, a ribonucleoproteinparticle (RNP) or a poloxamer. In some embodiments, a method ofintroducing polynucleotides to a cell using a non-viral vector deliverysystem includes electroporation, lipofection, microinjection,biolistics, or agent-enhanced uptake.

In some embodiments, a polynucleotide such as a gRNA, a sgRNA, an mRNAencoding a nuclease, or a donor template are delivered to a cell using aviral vector delivery system. Examples of a viral vector delivery systeminclude, but are not limited to, retroviral vectors, lentiviral vectors,adenovirus vectors, poxvirus vectors, herpesvirus vectors, andadeno-associated virus (AAV) vectors.

In some embodiments, a donor template encoding a CAR construct isdelivered to a cell as one or more polynucleotides. In some embodiments,a donor template encoding a CAR construct is delivered by a viraldelivery vehicle. In some embodiments, a viral delivery vehicle is anadeno-associated virus (AAV) vector.

In some embodiments, an endonuclease (e.g., Cas9) is delivered to a cellas a polypeptide. In some embodiments, an endonuclease (e.g., Cas9) isdelivered to a cell separately from a genome-targeting nucleic acid(e.g., a gRNA, a sgRNA). In some embodiments, an endonuclease (e.g.,Cas9) is delivered to a cell as a complex with one or moregenome-targeting polynucleotides (e.g., a gRNA, a sgRNA). In someembodiments, a endonuclease or a pre-complexed endonuclease is deliveredby a non-viral delivery vehicle that includes, but is not limited to, ananoparticle, a liposome, a ribonucleoprotein, a positively chargedpeptide, a small molecule RNA-conjugate, an aptamer-RNA chimeras, or anRNA-fusion protein complex. In some embodiments, a method of introducingan endonuclease polypeptide or a pre-complexed endonuclease polypeptideto a cell includes electroporation, lipofection, microinjection,biolistics, or agent-enhanced uptake.

In some embodiments, a Cas9 polypeptide is pre-complexed with one ormore sgRNAs to form a ribonucleoprotein particle (RNP). In someembodiments, a Cas9/sgRNA RNP is formulated using a lipid nanoparticle.In some embodiments, a donor template is formulated using an AAV vector.In some embodiments, delivery to a cell of a formulated Cas9/sgRNA RNPis performed by electroporation of the cell. In some embodiments, adonor template formulated as an AAV vector is delivered prior toelectroporation. In some embodiments, a donor template formulated as anAAV vector is delivered during electroporation. In some embodiments, adonor template formulated as an AAV vector is delivered followingelectroporation.

In some embodiments, a gene edit performed using a CRISPR/Cas9endonuclease results in an engineered T cell with a disrupted TRAC gene.In some embodiments, a disruption of a TRAC gene results in eliminatedor decreased expression of the TRAC gene. In some embodiments, adisruption of a TRAC gene disrupts or inhibits transcription andtranslation of an encoded gene product. In some embodiments, adisruption of a TRAC gene results in eliminated or decreased expressionof a TRAC gene product. In some embodiments, eliminated or decreasedexpression of the TRAC gene is associated with loss of function of theTCR. In some embodiments, loss of TCR function renders an engineered Tcell suitable to allogeneic transplantation (i.e., minimizing the riskof inducing GvHD). In some embodiments, a disruption of a TRAC gene iscreated by knocking in a CAR into the TRAC gene (e.g., using an AAVvector and a donor template). In some embodiments, a disruption in theTRAC gene expression is created by gRNAs targeting the TRAC genomicregion and knocking in a CAR into the CAR gene. In some embodiments, aknock-in CAR is provided by a donor template with homology arms thatcorrespond to sequences of the TRAC surrounding the site of a DSB.

In some embodiments, a gene edit performed using a CRISPR/Cas9endonuclease results in an engineered T cell with a disrupted β2M gene.In some embodiments, gRNAs targeting the B2M genomic region createindels in the β2M gene that disrupt or inhibit transcription andtranslation of an encoded gene product. In some embodiments, adisruption of a β2M gene results in eliminated or decreased expressionof the β2M polypeptide. In some embodiments, eliminated or decreasedexpression of the B2M polypeptide is associated with loss of function ofthe MHC I complex. In some embodiments, loss of MHC I function rendersan engineered T cell suitable to allogeneic transplantation (i e ,minimizing the risk of a host versus allogeneic T cell response). Insome embodiments, loss of MHC I function results in increasedpersistence of an engineered T cell in an allogeneic recipient.

In some embodiments, a gene edit performed using a CRISPR/Cas9endonuclease results in an engineered T cell with a disrupted CD70 gene.In some embodiments, gRNAs targeting the CD70 genomic region createindels in the CD70 gene that disrupt or inhibit transcription andtranslation of an encoded gene product. In some embodiments, adisruption of a CD70 gene results in eliminated or decreased expressionof the CD70 polypeptide. In some embodiments, eliminated or decreasedexpression of the CD70 polypeptide is associated with enhanced cellproliferation, enhanced in vivo persistence, decreased exhaustion,and/or enhanced anti-tumor efficacy.

In some embodiments, a gene edit performed using a CRISPR/Cas9endonuclease results in an engineered T cell with a disrupted PD-1 gene.In some embodiments, gRNAs targeting the PD-1 genomic region createindels in the PD-1 gene that disrupt or inhibit transcription andtranslation of an encoded gene product. In some embodiments, adisruption of a PD-1 gene results in eliminated or decreased expressionof the PD-1 polypeptide.

Methods and Compositions

Provided herein, in some embodiments, are methods for treating cancer.Non-limiting examples of cancers that may be treated as provided hereininclude multiple myeloma, leukemia (e.g., T cell leukemia, B-cell acutelymphoblastic leukemia (B-ALL), and/or chronic lymphocytic leukemia(C-CLL)), lymphoma (e.g., B-cell non-Hodgkin's lymphoma (B-NHL),Hodgkin's lymphoma, and/or T cell lymphoma), and/or clear cell renalcell carcinoma (ccRCC). In some embodiment, the methods comprisedelivering the CAR T cells (e.g., anti-BCMA, anti-CD19, anti-CD33 and/oranti-CD70 CAR T cells) of the present disclosure to a subject havingmultiple myeloma, leukemia, or lymphoma. Other non-limiting examples ofcancers (e.g., solid tumors) that may be treated as provided hereininclude pancreatic cancer, gastric cancer, ovarian cancer, cervicalcancer, breast cancer, renal cancer, thyroid cancer, nasopharyngealcancer, non-small cell lung (NSCLC), glioblastoma, and/or melanoma.

CD70 has also been detected on hematological tumors and on carcinomas.The restricted expression pattern of CD70 in normal tissues and itswidespread expression in various malignancies makes it an attractivetarget for antibody-based therapeutics. The use of CAR T cell therapy totarget CD70⁺ cancers, however, is potentially problematic because ofCD70 expression in the T cells. To address this potential problem, thepresent disclosure also provides CAR T cells that have been engineeredto disrupt endogenous CD70 expression while at the same time expressingan anti-CD70 binding moiety (e.g., an anti-CD70 scFv).

In some embodiments, the cancer is a CD70⁺ cancer. In other embodiments,the cancer is a BCMA⁺ cancer. In some embodiments, the cancer is aCD19+cancer. In some embodiments, the cancer is a CD33+ cancer. Itshould be understood that other cancers, expressing other cancerantigens, may be treated using the engineered CD70 knockout CAR T cellsof the present disclosure.

The methods, in some embodiments, comprise administering to a subject(e.g., a patient having a CD70⁺ cancer, a BCMA⁺ cancer, a CD19+ canceror a CD33+ cancer) a population of CAR T cells as provided herein. Insome embodiments, the methods comprise administering to a subject apopulation of CAR T cells comprising a CD70 gene knockout. In someembodiments, the methods comprise administering to a subject apopulation of CAR T cells comprising a CD70 gene knockout and a PD1 geneknockout. In some embodiments, the methods comprise implanting the cellsinto subject. This implanting step may be accomplished using any methodof implantation known in the art. For example, the engineered cells maybe injected directly in a subject's blood or otherwise administered tothe subject.

As demonstrated herein, CAR T cells comprising a CD70 gene knockoutexhibit extended proliferation and increased in vivo persistence. Insome embodiments, CAR T cells comprising a CD70 gene knockout exhibitincreased anti-tumor efficacy relative to CAR T cells expressingendogenous CD70. In some embodiments, CAR T cells comprising a CD70 geneknockout exhibit increased anti-tumor efficacy in solid tumors relativeto CAR T cells expressing endogenous CD70. Without wishing to be boundby theory, the increased in vivo persistence of CAR T cells comprising aCD70 gene knockout may allow for expansion in solid tumors and thereforeprovide enhanced anti-tumor efficacy in such tumors relative to CAR Tcells expressing endogenous CD70.

In some embodiments, the disclosure provides a method for treating asolid tumor with the CAR T cells described herein. In some embodiments,the disclosure provides a method for treating a solid tumor with theanti-CD70 CAR T cells described herein.

The step of administering may include the placement (e.g.,transplantation) of cells, e.g., engineered T cells, into a subject, bya method or route that results in at least partial localization of theintroduced cells at a desired site, such as tumor, such that a desiredeffect(s) is produced. Engineered T cells can be administered by anyappropriate route that results in delivery to a desired location in thesubject where at least a portion of the implanted cells or components ofthe cells remain viable. The period of viability of the cells afteradministration to a subject can be as short as a few hours, e.g.,twenty-four hours, to a few days, to as long as several years, or eventhe life time of the subject, i.e., long-term engraftment. For example,in some aspects described herein, an effective amount of engineered Tcells is administered via a systemic route of administration, such as anintraperitoneal or intravenous route.

A subject may be any subject for whom diagnosis, treatment, or therapyis desired. In some embodiments, the subject is a mammal. In someembodiments, the subject is a human.

A donor is an individual who is not the subject being treated. A donoris an individual who is not the patient. In some embodiments, a donor isan individual who does not have or is not suspected of having the cancerbeing treated. In some embodiments, multiple donors, e.g., two or moredonors, are used.

In some embodiments, an engineered T cell population being administeredaccording to the methods described herein comprises allogeneic T cellsobtained from one or more donors. Allogeneic refers to a cell, cellpopulation, or biological samples comprising cells, obtained from one ormore different donors of the same species, where the genes at one ormore loci are not identical to the recipient (e.g., subject). Forexample, an engineered T cell population, being administered to asubject can be derived from one or more unrelated donors, or from one ormore non-identical siblings. In some embodiments, syngeneic cellpopulations may be used, such as those obtained from geneticallyidentical donors, (e.g., identical twins). In some embodiments, thecells are autologous cells; that is, the engineered T cells are obtainedor isolated from a subject and administered to the same subject, i.e.,the donor and recipient are the same.

An effective amount refers to the amount of a population of engineered Tcells needed to prevent or alleviate at least one or more signs orsymptoms of a medical condition (e.g., cancer), and relates to asufficient amount of a composition to provide the desired effect, e.g.,to treat a subject having a medical condition. An effective amount alsoincludes an amount sufficient to prevent or delay the development of asymptom of the disease, alter the course of a symptom of the disease(for example but not limited to, slow the progression of a symptom ofthe disease), or reverse a symptom of the disease. It is understood thatfor any given case, an appropriate effective amount can be determined byone of ordinary skill in the art using routine experimentation.

For use in the various aspects described herein, an effective amount ofcells (e.g., engineered T cells) comprises at least 10² cells, at least5×10² cells, at least 10³ cells, at least 5×10³ cells, at least 10⁴cells, at least 5×10⁴ cells, at least 10⁵ cells, at least 2×10⁵ cells,at least 3×10⁵ cells, at least 4×10⁵ cells, at least 5×10⁵ cells, atleast 6×10⁵ cells, at least 7×10⁵ cells, at least 8×10⁵ cells, at least9×10⁵ cells, at least 1×10⁶ cells, at least 2×10⁶ cells, at least 3×10⁶cells, at least 4×10⁶ cells, at least 5×10⁶ cells, at least 6×10⁶ cells,at least 7×10⁶ cells, at least 8×10⁶ cells, at least 9×10⁶ cells, ormultiples thereof. The cells are derived from one or more donors, or areobtained from an autologous source. In some examples described herein,the cells are expanded in culture prior to administration to a subjectin need thereof.

Modes of administration include injection, infusion, instillation, oringestion. Injection includes, without limitation, intravenous,intramuscular, intra-arterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subarachnoid, intraspinal, intracerebro spinal, and intrasternalinjection and infusion. In some embodiments, the route is intravenous.

In some embodiments, engineered T cells are administered systemically,which refers to the administration of a population of cells other thandirectly into a target site, tissue, or organ, such that it enters,instead, the subject's circulatory system and, thus, is subject tometabolism and other like processes.

The efficacy of a treatment comprising a composition for the treatmentof a medical condition can be determined by the skilled clinician. Atreatment is considered “effective treatment,” if any one or all of thesigns or symptoms of, as but one example, levels of functional targetare altered in a beneficial manner (e.g., increased by at least 10%), orother clinically accepted symptoms or markers of disease (e.g., cancer)are improved or ameliorated. Efficacy can also be measured by failure ofa subject to worsen as assessed by hospitalization or need for medicalinterventions (e.g., progression of the disease is halted or at leastslowed). Methods of measuring these indicators are known to those ofskill in the art and/or described herein. Treatment includes anytreatment of a disease in subject and includes: (1) inhibiting thedisease, e.g., arresting, or slowing the progression of symptoms; or (2)relieving the disease, e.g., causing regression of symptoms; and (3)preventing or reducing the likelihood of the development of symptoms.

Combination therapies are also encompassed by the present disclosure.For example, CD70 and/or CD27 antibodies can be used to bind and/ormodulate the activity of CD70 and/or

CD27 on CAR T cells and promote a decrease in exhaustion, enhanced CAR Tcell expansion and increase efficacy of cancer cell killing. Thus, CD70and/or CD27 antibodies can be administered with any CAR T cell known inthe art to improve the CAR T cell function. For example, any of theengineered T cells provided herein may be administered in combinationwith anti-CD70 antibodies, anti-CD27 antibodies, or a combination ofanti-CD70 antibodies and anti-CD27 antibodies. In some embodiments,TRAC⁻/β2M⁻ CAR⁺ T cells (e.g., anti-CD70 CAR or anti-BCMA CAR) areadministered in combination with anti-CD70 and/or anti-CD27 antibodies.In some embodiments, TRAC⁻/β2M⁻/CD70⁻ CAR⁺ T cells (e.g., anti-CD70 CARor anti-BCMA CAR) are administered in combination with anti-CD70 and/oranti-CD27 antibodies. In some embodiments, TRAC⁻/β2M⁻/PD-1⁻/CAR⁺ T cells(e.g., anti-CD70 CAR or anti-BCMA CAR) are administered in combinationwith anti-CD70 and/or anti-CD27 antibodies. In some embodiments,TRAC⁻/β2M⁻/CD70⁻ CAR⁺ T cells (e.g., anti-CD70 CAR or anti-BCMA CAR) areadministered in combination with anti-CD70 and/or anti-CD27 antibodies.In some embodiments, the antibodies administered in combination can beVarlilumab.

In some embodiments, the disclosure provides a method of reducingexhaustion of T cells comprising disrupting the CD70 gene in the Tcells. In some embodiments, the disclosure provides a method ofincreasing proliferation of T cells comprising disrupting the CD70 genein the T cells. In some embodiments, the disclosure provides a method ofincreasing cytotoxicity of T cells comprising disrupting the CD70 genein the T cells. In some embodiments, the disclosure provides a method ofovercoming inhibitory effect of an immune checkpoint (e.g., PD-1) in Tcells comprising disrupting the CD70 gene in the T cells.

Other Embodiments

The disclosure relates to the following embodiments. Throughout thissection, the term embodiment is abbreviated as ‘E’ followed by anordinal. For example, E1 is equivalent to Embodiment 1.

-   E1. An engineered T cell comprising a disrupted CD70 gene, a    disrupted programmed cell death-1 (PD-1) gene, and a nucleic acid    encoding a chimeric antigen receptor (CAR).-   E2. An engineered T cell comprising a disrupted CD70 gene and a    nucleic acid encoding a chimeric antigen receptor (CAR) that binds    CD70.-   E3. An engineered T cell comprising a disrupted CD70 gene and a    nucleic acid encoding a chimeric antigen receptor (CAR) that does    not bind CD70.-   E4. The engineered T cell of embodiment 2 or 3, further comprising a    disrupted PD-1 gene.-   E5. The engineered T cell of any one of embodiments 1-4 further    comprising a disrupted T cell receptor alpha chain constant region    (TRAC) gene.-   E6. The engineered T cell of any one of embodiments 1-5 further    comprising a disrupted beta-2-microglobulin (β2M) gene.-   E7. An engineered T cell, comprising

a disrupted T cell receptor alpha chain constant region (TRAC) gene;

a disrupted beta-2-microglobulin (β2M) gene;

a disrupted CD70 gene; and

a nucleic acid encoding a chimeric antigen receptor (CAR).

-   E8. The engineered T cell of embodiment 7, wherein the nucleic acid    encoding the CAR is inserted into the TRAC gene.-   E9. The engineered T cell of embodiment 7 or 8, further comprising a    disrupted PD-1 gene.-   E10. The engineered T cell of any one of embodiments 1-9, wherein    the CAR comprises an ectodomain that comprises an anti-CD70    antibody, optionally wherein the anti-CD70 antibody is an anti-CD70    single-chain variable fragment (scFv).-   E11. The engineered T cell of embodiment 10, wherein the anti-CD70    scFv comprises the same heavy chain variable region (VH)    complementarity determining regions (CDRs) and the same light chain    variable region (VL) CDRs as a reference antibody, wherein the    reference antibody comprises a VH set forth as SEQ ID NO: 51 and a    VL set forth as SEQ ID NO: 52.-   E12. The engineered T cell of embodiment 11, wherein the anti-CD70    scFv comprises the same VH and VL chains as the reference antibody.-   E13. The engineered T cell of embodiment 11, wherein the anti-CD70    scFv comprises the amino acid sequence of SEQ ID NO: 48 or 50.-   E14. The engineered T cell of embodiment 11, wherein the anti-CD70    scFv comprises the amino acid sequence of SEQ ID NO: 50.-   E15. The engineered T cell of any one of embodiments 1-9, wherein    the CAR comprises an ectodomain that comprises an anti-BCMA    antibody, optionally wherein the anti-BCMA antibody is an anti-BCMA    single-chain variable fragment (scFv).-   E16. The engineered T cell of embodiment 15, wherein the anti-BCMA    scFv comprises the same VH complementarity determining regions    (CDRs) and the same VL CDRs as a reference antibody, wherein the    reference antibody comprises a VH) set forth as SEQ ID NO: 60 and a    VL set forth as SEQ ID NO: 61.-   E17. The engineered T cell of embodiment 16, wherein the anti-BCMA    scFv comprises the same VH and VL chains as the reference antibody.-   E18. The engineered T cell of embodiment 16, wherein the anti-BCMA    scFv comprises the amino acid sequence of SEQ ID NO: 59.-   E19. The engineered T cell of any one of embodiments 1-18, wherein    the CAR comprises a CD28 or 41BB co-stimulatory domain and    optionally a CD3t signaling domain.-   E20. The engineered T cell of any one of embodiments 5-19, wherein    the TRAC gene comprises the nucleotide sequence of SEQ ID NO: 44 or    55 and/or the nucleic acid encoding the CAR comprises the nucleotide    sequence of SEQ ID NO: 45 or 56.-   E21. The engineered T cell of any one of embodiments 6-20, wherein    the disrupted β2M gene comprises gene at least one nucleotide    sequence selected from any one of SEQ ID NOS: 9-14.-   E22. The engineered T cell of any one of embodiments 1-21, wherein    the engineered T cell maintains cytotoxicity following 5    rechallenges with a cancer cell.-   E23. The engineered T cell of embodiment 22, wherein the engineered    T cell maintains cytotoxicity following 10 rechallenges with a    cancer cell.-   E24. A population of cells comprising engineered T cells that    comprise a disrupted CD70 gene, a disrupted programmed cell death-1    (PD-1) gene, and a nucleic acid encoding a chimeric antigen receptor    (CAR).-   E25. A population of cells comprising engineered T cells that    comprise a disrupted CD70 gene, and a nucleic acid encoding a    chimeric antigen receptor (CAR) that binds CD70.-   E26. A population of cells comprising engineered T cells that    comprise a disrupted CD70 gene and a nucleic acid encoding a    chimeric antigen receptor (CAR) that does not bind CD70.-   E27. The population of cells of embodiment 25 or 26 further    comprising a disrupted programmed cell death-1 (PD-1) gene.-   E28. The population of cells of any one of embodiments 24-27 further    comprising a disrupted T cell receptor alpha chain constant region    (TRAC) gene.-   E29. The population of cells of any one of embodiments 24-28 further    comprising a disrupted beta-2-microglobulin (β2M) gene.-   E30. A population of cells comprising

engineered T cells that comprise

a disrupted T cell receptor alpha chain constant region (TRAC) gene;

a disrupted beta-2-microglobulin (β2M) gene;

a disrupted CD70 gene; and

a nucleic acid encoding a chimeric antigen receptor (CAR).

-   E31. The population of cells of embodiment 30, wherein the nucleic    acid encoding the CAR is inserted into the TRAC gene.-   E32. The population of cells of embodiment 30 or 31, wherein the    engineered T cells further comprise a disrupted programmed cell    death-1 (PD-1) gene.-   E33. The population of cells of any one of embodiments 24 or 27-32,    wherein the CAR comprises an ectodomain that comprises an anti-CD70    antibody, optionally wherein the anti-CD70 antibody is an anti-CD70    single-chain variable fragment (scFv).-   E34. The population of cells of embodiment 33, wherein the anti-CD70    scFv comprises the same VH complementarity determining regions    (CDRs) and the same VL CDRs as a reference antibody, wherein the    reference antibody comprises a VH set forth as SEQ ID NO: 51 and a    VL set forth as SEQ ID NO: 52.-   E35. The population of cells of embodiment 34, wherein the anti-CD70    scFv comprises the same VH and VL chains as the reference antibody.-   E36. The population of cells of embodiment 35, wherein the anti-CD70    scFv comprises the amino acid sequence of SEQ ID NO: 48 or 50.-   E37. The population of cells of embodiment 35, wherein the anti-CD70    scFv comprises the amino acid sequence of SEQ ID NO: 50.-   E38. The population of cells of any one of embodiments 24-32,    wherein the CAR comprises an ectodomain that comprises an anti-BCMA    antibody, optionally wherein the anti-BCMA antibody is an anti-BCMA    single-chain variable fragment (scFv).-   E39. The population of cells of embodiment 38, wherein the anti-BCMA    scFv comprises the same VH complementarity determining regions    (CDRs) and the same VL CDRs as a reference antibody, wherein the    reference antibody comprises a VH set forth as SEQ ID NO: 60 and a    VL set forth as SEQ ID NO: 61.-   E40. The population of cells of embodiment 39, wherein the anti-BCMA    scFv comprises the same VH and VL chains as the reference antibody.-   E41. The population of cells of embodiment 39, wherein the anti-BCMA    scFv comprises the amino acid sequence of SEQ ID NO: 59.-   E42. The population of cells of any one of embodiments 28-41,    wherein the TRAC gene comprises the nucleotide sequence of SEQ ID    NO: 44 or 55 and/or the nucleic acid encoding the CAR comprises the    nucleotide sequence of SEQ ID NO: 45 or 56.-   E43. The population of cells of any one of embodiments 29-42,    wherein the disrupted β2M gene comprises gene at least one    nucleotide sequence selected from any one of SEQ ID NOS: 9-14.-   E44. The population of cells of any one of embodiments 24-43,    wherein at least 50% of the engineered T cells do not express a    detectable level of TCR surface protein, do not express a detectable    level of β2M surface protein, do not express a detectable level of    CD70 surface protein, do not express a detectable level of PD-1    surface protein, and/or express a detectable level of the CAR.-   E45. The population of cells of embodiment 44, wherein 50%-70%, of    the engineered T cells do not express a detectable level of TCR    surface protein, do not express a detectable level of β2M surface    protein, do not express a detectable level of CD70 surface protein,    do not express a detectable level of PD-1 surface protein, and/or    express a detectable level of the CAR.-   E46. The population of cells of any one of embodiments 28-45,    wherein at least 90%, optionally 90%-100%, of the engineered T cells    do not express a detectable level of TCR surface protein.-   E47. The population of cells of any one of embodiments 29-46,    wherein at least 60%, optionally 60%-75%, of the engineered T cells    do not express a detectable level of β2M surface protein.-   E48. The population of cells of any one of embodiments 24-47,    wherein at least 80%, optionally 80%-100%, of the engineered T cells    do not express a detectable level of CD70 surface protein.-   E49. The population of cells of any one of embodiments 1-48, wherein    at least 80%, optionally 80%-95%, of the engineered T cells express    a detectable level of the CAR.-   E50. The population of cells of any one of embodiments 24-49,    wherein the engineered T cells exhibit at least 20% greater cellular    proliferative capacity, relative to control T cells.-   E51. The population of cells of any one of embodiments 24-50,    wherein the engineered T cells exhibit at least 20% greater cellular    lysis capability, relative to control T cells.-   E52. The population of cells of any one of embodiments 24-51,    wherein the level of cytokines secreted by the engineered T cells    are at least 2-fold greater than the level of cytokines secreted by    control T cells.-   E53. The population of any one of embodiments 24-52, wherein the    engineered T cells exhibit reduced cellular exhaustion, relative to    control T cells.-   E54. The population of cells of embodiment 53, wherein the    engineered T cells express reduced levels of LAG3, relative to    control T cells.-   E55. The population of cells of any one of embodiments 54, wherein    the control T cells are engineered T cells that express endogenous    CD70 protein.-   E56. The population of cells of any one of embodiments 24-55,    wherein the engineered T cells maintain cytokine-dependent    proliferation.-   E57. The population of cells of any one of embodiments 24-56,    wherein the engineered T cells maintain cytotoxicity following 5    rechallenges with a cancer cell.-   E58. The population of cells of embodiment 47, wherein the    engineered T cells maintain cytotoxicity following 10 rechallenges    with a cancer cell.-   E59. A method comprising administering to a subject the population    of cells of any one of embodiments 24-58.-   E60. The method of embodiment 59, wherein the engineered T cells are    engineered human T cells.-   E61. The method of embodiment 59 or 60, wherein the subject has a    cancer.-   E62. The method of embodiment 61, wherein the cancer expresses CD70    and/or BCMA.-   E63. The method of any one of embodiments 59-62, wherein the    population of cells is administered to the subject in an amount    effective to treat the cancer.-   E64. The method of any one of embodiments 59-63, wherein the cancer    is a solid tumor malignancy or a hematological malignancy.-   E65. The method embodiment 64, wherein the solid tumor malignancy is    selected from the group consisting of: ovarian tumor, pancreatic    tumor, kidney tumor, lung tumor, and intestinal tumor.-   E66. The method of embodiment 63, wherein the population of cells is    administered to the subject in an amount effective to reduce the    volume of a tumor in the subject.-   E67. A method for producing an engineered T cell, the method    comprising

(a) delivering to a T cell

-   -   -   an RNA-guided nuclease,        -   a gRNA targeting a CD70 gene, and        -   a vector comprising a donor template that comprises a            nucleic acid encoding a CAR; and

(b) producing an engineered T cell comprising a disrupted CD70 gene andexpressing the CAR.

-   E68. The method of embodiment 67, further comprising in step (a)    delivering to the T cell a gRNA targeting a PD-1 gene; wherein the    engineered T cell of step (b) further comprises a disrupted PD-1    gene.-   E69. The method of embodiment 67 or embodiment 68, further    comprising in step (a) delivering to the T cell a gRNA targeting a    TRAC gene; wherein the engineered T cell of step (b) further    comprises a disrupted TRAC gene.-   E70. The method of embodiment 69, wherein the nucleic acid encoding    the CAR is flanked by left and right homology arms to the TRAC gene    locus; and wherein the engineered T cell of step (b) comprises the    nucleic acid encoding the CAR inserted into the TRAC gene locus.-   E71. The method of any one of embodiments embodiment 67-70, further    comprising in step

(a) delivering to the T cell a gRNA targeting a β2M gene; wherein theengineered T cell of step

(b) further comprises a disrupted β2M gene.

-   E72. A method for producing an engineered T cell, the method    comprising

(a) delivering to a T cell

an RNA-guided nuclease,

a gRNA targeting a TRAC gene,

a gRNA targeting a β2M gene,

a gRNA targeting a CD70 gene, and

a vector comprising a donor template that comprises a nucleic acidencoding a CAR; and

(b) producing an engineered T cell.

-   E73. The method of embodiment 72, wherein the nucleic acid encoding    the CAR is flanked by left and right homology arms to the TRAC gene    locus.-   E74. The method of embodiment 72 or 73 further comprising delivering    to the T cell a gRNA targeting a PD-1 gene.-   E75. The method of any one of embodiments 67-74, wherein the    RNA-guided nuclease is a Cas9 nuclease, optionally a S. pyogenes    Cas9 nuclease.-   E76. The method of any one of embodiments 69-75, wherein the gRNA    targeting the TRAC gene comprises the nucleotide sequence of SEQ ID    NO: 98 or targets the nucleotide sequence of SEQ ID NO: 118, and    optionally wherein the gRNA targeting the TRAC gene comprises the    nucleotide sequence of SEQ ID NO: 30.-   E77. The method of any one of embodiments 71-76, wherein the gRNA    targeting the β2M gene comprises the nucleotide sequence of SEQ ID    NO: 99 or targets the nucleotide sequence of SEQ ID NO: 119, and    optionally wherein the gRNA targeting the β2M gene comprises the    nucleotide sequence of SEQ ID NO: 31.-   E78. The method of any one of embodiments 67-77, wherein the gRNA    targeting the CD70 gene comprises the nucleotide sequence of SEQ ID    NOS: 94 or 95 or targets the nucleotide sequence of SEQ ID NO: 114    or 115, and optionally wherein the gRNA targeting the CD70 gene    comprises the nucleotide sequence of SEQ ID NOS: 26 or 27.-   E79. The method of any one of embodiments 68-71 and 74-78, wherein    the gRNA targeting the PD-1 gene comprises the nucleotide sequence    of SEQ ID NO: 100 or targets the nucleotide sequence of SEQ ID NO:    120, and optionally wherein the gRNA targeting the PD-1 gene    comprises the nucleotide sequence of SEQ ID NO: 32.-   E80. The method of any one of embodiments 67-79, wherein the CAR    comprises an ectodomain that comprises an anti-CD70 antibody,    optionally wherein the anti-CD70 antibody is an anti-CD70    single-chain variable fragment (scFv).-   E81. The method of embodiment 80, wherein the anti-CD70 scFv    comprises the same VH complementarity determining regions (CDRs) and    the same VL CDRs as a reference antibody, wherein the reference    antibody comprises a VH set forth as SEQ ID NO: 51 and a VL set    forth as SEQ ID NO: 52.-   E82. The method of embodiment 81, wherein the anti-CD70 scFv    comprises the same VH and VL chains as the reference antibody.-   E83. The method of embodiment 81, wherein the anti-CD70 scFv    comprises the amino acid sequence of SEQ ID NO: 48 or 50.-   E84. The method of embodiment 81, wherein the anti-CD70 scFv    comprises the amino acid sequence of SEQ ID NO: 50.-   E85. The method of any one of embodiments 67-79, wherein the CAR    comprises an ectodomain that comprises an anti-BCMA antibody,    optionally wherein the anti-BCMA antibody is an anti-BCMA    single-chain variable fragment (scFv).-   E86. The method of embodiment 85, wherein the anti-BCMA scFv    comprises the same VH complementarity determining regions (CDRs) and    the same VL CDRs as a reference antibody, wherein the reference    antibody comprises a VH set forth as SEQ ID NO: 60 and a VL set    forth as SEQ ID NO: 61.-   E87. The method of embodiment 86, wherein the anti-BCMA scFv    comprises the same VH and VL chains as the reference antibody.-   E88. The method of embodiment 86, wherein the anti-BCMA scFv    comprises the amino acid sequence of SEQ ID NO: 59.-   E89. The method of any one of embodiments 67-88, wherein the CAR    further comprises a CD28 or 41BB co-stimulatory domain and    optionally a CD3z signaling domain.-   E90. The method of embodiment 72, wherein the vector comprises a    nucleic acid encoding a CAR that comprises the amino acid sequence    of SEQ ID NO: 46.-   E91. The method of embodiment 72, wherein the vector comprises a    nucleic acid encoding a CAR that comprises the amino acid sequence    of SEQ ID NO: 57.-   E92. An engineered T cell comprising an RNA-guided nuclease and a    gRNA targeting a CD70 gene, optionally wherein the gRNA targeting    the CD70 gene comprises the nucleotide sequence of SEQ ID NOS: 94 or    95 or targets the nucleotide sequence of SEQ ID NO: 114 or 115, and    optionally wherein the gRNA targeting the CD70 gene comprises the    nucleotide sequence of SEQ ID NOS: 26 or 27.-   E93. The engineered T cell of embodiment 92 further comprising a    gRNA targeting a PD-1 gene, optionally wherein the gRNA targeting    the PD-1 gene comprises the nucleotide sequence of SEQ ID NO: 100 or    targets the nucleotide sequence of SEQ ID NO: 120, and optionally    wherein the gRNA targeting the PD-1 gene comprises the nucleotide    sequence of SEQ ID NO: 32.-   E94. The engineered T cell of embodiment 92 or 93 further comprising    a gRNA targeting a TRAC gene, optionally wherein the gRNA targeting    the TRAC gene comprises the nucleotide sequence of SEQ ID NO: 98 or    targets the nucleotide sequence of SEQ ID NO: 118, and optionally    wherein the gRNA targeting the TRAC gene comprises the nucleotide    sequence of SEQ ID NO: 30.-   E95. The engineered T cell of any one of embodiments 92-94 further    comprising a gRNA targeting a β2M gene, optionally wherein the gRNA    targeting the β2M gene comprises the nucleotide sequence of SEQ ID    NO: 99 or targets the nucleotide sequence of SEQ ID NO: 119, and    optionally wherein the gRNA targeting the β2M gene comprises the    nucleotide sequence of SEQ ID NO: 31.-   E96. The engineered T cell of any one of embodiments 92-95, wherein    the RNA-guided nuclease is a Cas9 nuclease, optionally a S. pyogenes    Cas9 nuclease.-   E97. The engineered T cell of any one of embodiments 92-96 further    comprising a vector comprising a donor template that comprises a    nucleic acid encoding a CAR, optionally wherein the nucleic acid    encoding the CAR is flanked by left and right homology arms to the    TRAC gene locus.-   E98. The engineered T cell of embodiment 97, wherein the CAR    comprises an ectodomain that comprises an anti-CD70 antibody,    optionally wherein the anti-CD70 antibody is an anti-CD70    single-chain variable fragment (scFv).-   E99. The engineered T cell of embodiment 98, wherein the anti-CD70    scFv comprises the same VH complementarity determining regions    (CDRs) and the same VL CDRs as a reference antibody, wherein the    reference antibody comprises a VH set forth as SEQ ID NO: 51 and a    VL set forth as SEQ ID NO: 52.-   E100. The engineered T cell of embodiment 99, wherein the anti-CD70    scFv comprises the same VH and VL chains as the reference antibody.-   E101. The engineered T cell of embodiment 99, wherein the anti-CD70    scFv comprises the amino acid sequence of SEQ ID NO: 48 or 50.-   E102. The engineered T cell of embodiment 99, wherein the anti-CD70    scFv comprises the amino acid sequence of SEQ ID NO: 50.-   E103. The engineered T cell of embodiment 97 wherein the CAR    comprises an ectodomain that comprises an anti-BCMA antibody,    optionally wherein the anti-BCMA antibody is an anti-BCMA    single-chain variable fragment (scFv).-   E104. The engineered T cell of embodiment 103, wherein the anti-BCMA    scFv comprises the same VH complementarity determining regions    (CDRs) and the same VL CDRs as a reference antibody, wherein the    reference antibody comprises a VH set forth as SEQ ID NO: 60 and a    VL set forth as SEQ ID NO: 61.-   E105. The engineered T cell of embodiment 104, wherein the anti-BCMA    scFv comprises the same VH and VL chains as the reference antibody.-   E106. The engineered T cell of embodiment 104, wherein the anti-BCMA    scFv comprises the amino acid sequence of SEQ ID NO: 59.-   E107. The engineered T cell of embodiment 97, wherein the vector    comprises a nucleic acid encoding a CAR that comprises the amino    acid sequence of SEQ ID NO: 46 or 57.-   E108. A method of increasing proliferation or reducing exhaustion of    T cells, the method comprising disrupting the CD70 gene in the T    cells.-   E109. The method of embodiment 108 further comprising disrupting in    the T cells at least one gene selected from the group consisting of:    programmed cell death-1 (PD-1) gene, T cell receptor alpha chain    constant region (TRAC) gene, and beta-2-microglobulin (β2M) gene.-   E110. The method of any one of embodiments 108-109 further    comprising expressing in the T cells a nucleic acid encoding a    chimeric antigen receptor (CAR).-   E111. The method of any one of embodiments 108-110, wherein the CD70    gene is disrupted by CRISPR/Cas gene editing.-   E112. The method of any one of embodiments 110-111, wherein the    PD-1, TRAC, and/or β2M gene is disrupted by CRISPR/Cas gene editing.-   E113. A method for treating cancer in a subject, comprising    administering to the patient a population of cells comprising    engineered T cells, wherein the engineered T cells comprise a    disrupted CD70 gene and a nucleic acid encoding a CAR, thereby    treating cancer in the subject.-   114. The method of embodiment 113, wherein the CAR binds CD70.-   E115. The method of embodiment 113, wherein the CAR does not bind    CD70.-   E116. The method of any one of embodiments 113-115, wherein the    engineered T cells further comprise a disrupted TRAC gene.-   E117. The method of any one of embodiments 113-116, wherein the    engineered T cells further comprise a disrupted B2M gene.-   E118. The method of any one of embodiments 113-116, wherein the    engineered T cells further comprise a disrupted PD-1 gene.-   E119. A method for treating cancer in a subject, comprising    administering to the patient a population of cells comprising    engineered T cells, wherein the engineered T cells comprise:

(i) a disrupted TRAC gene;;

(ii) a disrupted B2M gene;

(iii) a disrupted CD70 gene; and

(iv) a nucleic acid encoding a CAR;

thereby treating the cancer in the subject.

-   E120. The method of any one of embodiments 113-114 and 116-119,    wherein the CAR comprises (a) an ectodomain that comprises an    anti-CD70 antigen-binding fragment, (b) a CD8 transmembrane domain,    and (c) an endodomain that comprises a 41BB co-stimulatory domain    and a CD3z co-stimulatory domain.-   E121. The method of embodiment 119 or 120, wherein the disrupted    TRAC gene comprises the nucleic acid encoding the CAR.-   E122. The method of any one of embodiments 120-121, wherein the    anti-CD70 antibody is an anti-CD70 scFv.-   E123. The method of embodiment 122, wherein the anti-CD70 scFv    comprises the same heavy chain variable region (VH) complementarity    determining regions (CDRs) and the same light chain variable region    (VL) CDRs as a reference antibody, wherein the reference antibody    comprises a VH set forth as SEQ ID NO: 51 and a VL set forth as SEQ    ID NO: 52.-   E124. The method of embodiment 123, wherein the anti-CD70 scFv    comprises the same VH and VL chains as the reference antibody.-   E125. The method of embodiment 122, wherein the anti-CD70 scFv    comprises the amino acid sequence of SEQ ID NO: 48 or 50.-   E126. The method of embodiment 122, wherein the anti-CD70 scFv    comprises the amino acid sequence of SEQ ID NO: 50.-   E127. The method of any one of embodiments 113 and 115-118, wherein    the CAR comprises an ectodomain that comprises an anti-BCMA    antibody, optionally wherein the anti-BCMA antibody is an anti-BCMA    single-chain variable fragment (scFv).-   E128. The method of embodiment 127, wherein the anti-BCMA scFv    comprises the same VH complementarity determining regions (CDRs) and    the same VL CDRs as a reference antibody, wherein the reference    antibody comprises a VH) set forth as SEQ ID NO: 60 and a VL set    forth as SEQ ID NO: 61.-   E129. The method of embodiment 128, wherein the anti-BCMA scFv    comprises the same VH and VL chains as the reference antibody.-   E130. The method of embodiment 127, wherein the anti-BCMA scFv    comprises the amino acid sequence of SEQ ID NO: 59.-   E131. The method of any one of embodiments 113-130, wherein the    engineered T cells are engineered human T cells.-   E132. The method of any one of embodiments 113-131, wherein the    cancer expresses CD70 and/or BCMA.-   E133. The method of any one of embodiments 113-132, wherein the    population of cells is administered to the subject in an amount    effective to treat the cancer.-   E134. The method of any one of embodiments 113-133, wherein the    cancer is a solid tumor malignancy or a hematological malignancy.-   E135. The method embodiment 134, wherein the solid tumor malignancy    is selected from the group consisting of: ovarian tumor, pancreatic    tumor, kidney tumor, lung tumor, and intestinal tumor.-   E136. A population of cells comprising engineered T cells, wherein    the engineered T cells comprise:

(i) a disrupted TRAC gene;;

(ii) a disrupted beta-2-microglobulin (B2M) gene;

(iii) a disrupted CD70 gene

(iv) a nucleic acid encoding a CAR comprising (a) an ectodomain thatcomprises an anti-CD70 antigen-binding fragment, (b) a CD8 transmembranedomain, and (c) an endodomain that comprises a 41BB co-stimulatorydomain and a CD3z co-stimulatory domain.

-   E137. The population of cells of embodiment 136, wherein the    disrupted TRAC gene comprises the nucleic acid encoding the CAR.-   E138. The population of cells of any one of embodiments 136-137,    wherein the engineered T cells are human T cells.-   E139. An engineered T cell comprising:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises anucleic acid encoding a CAR comprising the amino acid sequence set forthin SEQ ID NO: 46;

(ii) a disrupted B2M gene; and

(iii) a disrupted CD70 gene.

-   E140. The engineered T cell of embodiment 139, wherein the nucleic    acid encoding the CAR comprises a sequence at least 80%, at least    90%, at least 95%, at least 96%, at least 97%, at least 98% or at    least 99% identical to SEQ ID NO: 45.-   E141. An engineered T cell comprising:

(i) a disrupted TRAC gene, wherein the disrupted TRAC gene comprises anucleic acid encoding a CAR, wherein the nucleic acid sequence is atleast 90% identical to SEQ ID NO: 45;

(ii) a disrupted B2M gene; and

(iii) a disrupted CD70 gene.

-   E142. The engineered T cell of any one of embodiments 139-141,    wherein the disrupted TRAC gene comprises a donor sequence    comprising the nucleotide sequence set forth in SEQ ID NO: 45 or SEQ    ID NO: 44.-   E143. An engineered T cell comprising:

(i) a disrupted TRAC gene comprising the nucleic acid sequence of SEQ IDNO: 44;

(ii) a disrupted B2M gene; and

(iii) a disrupted CD70 gene.

-   E144. The engineered T cell of any one of embodiments 139-143,    wherein the T cell is a human T cell.

EXAMPLES Example 1. Efficient Knockout of CD70 by Cas9:sgRNA RNPs in TCells

This example describes efficient editing of the CD70 gene in primaryhuman T cells ex vivo using CRISPR/Cas9 gene editing. Genomic segmentsof the CD70 gene containing the first three (3) protein coding exonswere used as input in gRNA design software. The genomic segments alsoincluded flanking splice site acceptor/donor sequences. Desired gRNAswere those that would lead to insertions or deletions in the codingsequence, disrupting the amino acid sequence of CD70, leading to out offrame/loss of function allele(s) (referred to as “CD70 knockout”alleles). All seven (7) in silico-identified gRNA spacer sequencestargeting the CD70 gene were synthesized, and the gRNAs werespecifically modified, as indicated in Table 5. While the gRNAs in Table5 were modified with 2′-O-methyl phosphorothioate modifications,unmodified gRNAs, or gRNAs with other modifications, may be used. Seealso PCT/IB2018/001619, filed May 11, 2018, herein incorporated in itsentirety by this reference.

TABLE 5 CD70 gRNA Sequences/Target Sequences gRNA Sequences NameUnmodified Sequence Modified Sequence CD70 sgRNA (E1_T1)UCACCAAGCCCGCGACCAAUg U*C*A*CCAAGCCCGCGACCAuuuuagagcuagaaauagcaaguuaaaaua AUguuuuagagcuagaaauagcaaguuaaaggcuaguccguuaucaacuugaaaaagug aauaaggcuaguccguuaucaacuugaaaagcaccgagucggugcUUUU aguggcaccgagucggugcU*U*U*U (SEQ ID NO: 23)(SEQ ID NO: 33) CD70 sgRNA (E1_T1) spacer UCACCAAGCCCGCGACCAAUU*C*A*CCAAGCCCGCGACCA (SEQ ID NO: 91) AU (SEQ ID NO: 101)CD70 sgRNA (E1_T3) AUCACCAAGCCCGCGACCAAg A*U*C*ACCAAGCCCGCGACCuuuuagagcuagaaauagcaaguuaaaaua AAguuuuagagcuagaaauagcaaguuaaaggcuaguccguuaucaacuugaaaaagug aauaaggcuaguccguuaucaacuugaaaagcaccgagucggugcUUUU aguggcaccgagucggugcU*U*U*U (SEQ ID NO: 24)(SEQ ID NO: 34) CD70 sgRNA (E1_T3) spacer AUCACCAAGCCCGCGACCAAA*U*C*ACCAAGCCCGCGACC (SEQ ID NO: 92) AA (SEQ ID NO: 102)CD70 sgRNA (E1_T4) CGGUGCGGCGCAGGCCCUAU C*G*G*UGCGGCGCAGGCCCUguuuuagagcuagaaauagcaaguuaaaau AUguuuuagagcuagaaauagcaaguuaaaaggcuaguccguuaucaacuugaaaaagu aauaaggcuaguccguuaucaacuugaaaaggcaccgagucggugcUUUU aguggcaccgagucggugcU*U*U*U (SEQ ID NO: 25)(SEQ ID NO: 35) CD70 sgRNA (E1_T4) spacer CGGUGCGGCGCAGGCCCUAUC*G*G*UGCGGCGCAGGCCCU (SEQ ID NO: 93) AU (SEQ ID NO: 103)CD70 sgRNA (E1_T7)); also GCUUUGGUCCCAUUGGUCGC G*C*U*UUGGUCCCAUUGGUCreferred to as: T7 guuuuagagcuagaaauagcaaguuaaaauGCguuuuagagcuagaaauagcaaguuaa aaggcuaguccguuaucaacuugaaaaaguaauaaggcuaguccguuaucaacuugaaaa ggcaccgagucggugcUUUUaguggcaccgagucggugcU*U*U*U (SEQ ID NO: 26) (SEQ ID NO: 36)CD70 sgRNA (E1_T7) spacer GCUUUGGUCCCAUUGGUCGC G*C*U*UUGGUCCCAUUGGUC(SEQ ID NO: 94) GC (SEQ ID NO: 104) CD70 sgRNA (E1_T8); alsoGCCCGCAGGACGCACCCAUAg G*C*C*CGCAGGACGCACCCA referred to as: T8uuuuagagcuagaaauagcaaguuaaaaua UAguuuuagagcuagaaauagcaaguuaaaggcuaguccguuaucaacuugaaaaagug aauaaggcuaguccguuaucaacuugaaaagcaccgagucggugcUUUU aguggcaccgagucggugcU*U*U*U (SEQ ID NO: 27)(SEQ ID NO: 37) CD70 sgRNA (E1_T8) spacer GCCCGCAGGACGCACCCAUAG*C*C*CGCAGGACGCACCCA (SEQ ID NO: 95) UA (SEQ ID NO: 105)CD70 sgRNA (E1_T10) GUGCAUCCAGCGCUUCGCAC G*U*G*CAUCCAGCGCUUCGCguuuuagagcuagaaauagcaaguuaaaau ACguuuuagagcuagaaauagcaaguuaaaaggcuaguccguuaucaacuugaaaaagu aauaaggcuaguccguuaucaacuugaaaaggcaccgagucggugcUUUU aguggcaccgagucggugcU*U*U*U (SEQ ID NO: 28)(SEQ ID NO: 38) CD70 sgRNA (E1_T10) spacer GUGCAUCCAGCGCUUCGCACG*U*G*CAUCCAGCGCUUCGC (SEQ ID NO: 96) AC (SEQ ID NO: 106)CD70 sgRNA (E3_T1) CAGCUACGUAUCCAUCGUGA C*A*G*CUACGUAUCCAUCGUguuuuagagcuagaaauagcaaguuaaaau GAguuuuagagcuagaaauagcaaguuaaaaggcuaguccguuaucaacuugaaaaagu aauaaggcuaguccguuaucaacuugaaaaggcaccgagucggugcUUUU aguggcaccgagucggugcU*U*U*U (SEQ ID NO: 29)(SEQ ID NO: 39) CD70 sgRNA (E3_T1) spacer CAGCUACGUAUCCAUCGUGAC*A*G*CUACGUAUCCAUCGU (SEQ ID NO: 97) GA (SEQ ID NO: 107)Target Sequences Name Target Sequence (PAM) CD70 sgRNA (E1_T1)TCACCAAGCCCGCGACCAAT (GGG) (SEQ ID NO: 111) CD70 sgRNA (E1_T3)ATCACCAAGCCCGCGACCAA (TGG) (SEQ ID NO: 112) CD70 sgRNA (E1_T4)CGGTGCGGCGCAGGCCCTAT (GGG) (SEQ ID NO: 113) CD70 sgRNA (E1_T7)GCTTTGGTCCCATTGGTCGC (GGG) (SEQ ID NO: 114) CD70 sgRNA (E1_T8)GCCCGCAGGACGCACCCATA (GGG) (SEQ ID NO: 115) CD70 sgRNA (E1_T10)GTGCATCCAGCGCTTCGCAC (AGG) (SEQ ID NO: 116) CD70 sgRNA (E3_T1)CAGCTACGTATCCATCGTGA (TGG) (SEQ ID NO: 117) TRAC sgRNAAGAGCAACAGTGCTGTGGCC (TGG) (SEQ ID NO: 118) β2M sgRNAGCTACTCTCTCTTTCTGGCC (TGG) (SEQ ID NO: 119) PD-1 sgRNACTGCAGCTTCTCCAACACAT (CGG) (SEQ ID NO: 120) *: 2′-O-methylphosphorothioate residue

Primary human T cells were transfected (electroporated) with aribonucleoprotein particle (RNP) containing Cas9 nuclease and asynthetic modified sgRNA targeting the CD70 gene (sequences in Table 5)or controls (no Cas9, no gRNA). Four to six (4-6) days posttransfection, cells were (1) subjected to a TIDE analysis to assessindel frequency and (2) processed by flow cytometry (primary antibody:FITC anti-human CD70 antibody, clone 113-16, Biolegend) to assess CD70expression levels at the cell surface.

Seven (7) gRNAs yielded measurable data by TIDE analysis, as indicatedin Table 6. Four (4) gRNA sequences yielded indel percentages (editingfrequencies) above 85% with protein expression knockdown above 80% (SEQID NOS: 23, 26, 27 and 29), indicating highly efficient gene editing.The data in Table 6 are from one (1) donor. The level of CD70 proteinexpression (assessed by median fluorescent intensity (MFI)) per testsample was normalized to the level of CD70 protein expression present incontrol cells.

TABLE 6 CD70 gRNA sequences, cutting efficiencies, and CD70 surfaceprotein expression in gene edited T cells Protein expression knockdowngRNA Name gRNA Spacer Sequence Indel % R² % CD70 EXON1_T1 (E1_T1)UCACCAAGCCCGCGACCAAU 89.3% 0.97 84.8% (SEQ ID NO: 91)CD70 EXON1_T3 (E1_T3) AUCACCAAGCCCGCGACCAA 65.2% 0.93 84.0%(SEQ ID NO: 92) CD70 EXON1_T4 (E1_T4) CGGUGCGGCGCAGGCCCUAU 81.6% 0.8387.5% (SEQ ID NO: 93) CD70 EXON1_T7 (E1_T7) GCUUUGGUCCCAUUGGUCGC 97.8%0.98 87.7% (SEQ ID NO: 94) CD70 EXON1_T8 (E1_T8) GCCCGCAGGACGCACCCAUA90.1% 0.94 88.1% (SEQ ID NO: 95) CD70 EXON1_T10 (E1_T10)GUGCAUCCAGCGCUUCGCAC 28.3% 0.30 83.9% (SEQ ID NO: 96)CD70 EXON3_T1 (E3_T1) CAGCUACGUAUCCAUCGUGA 85.6% 0.93 87.2%(SEQ ID NO: 97)

Analysis of On-Target Indel Profiles in T Cells

On-target amplicon analysis was conducted at the CD70 locus followinggene editing using the T7 guide (SEQ ID NO: 26; SEQ ID NO: 36),targeting the CD70 gene: GCTTTGGTCCCATTGGTCGC (SEQ ID NO: 160; targetsequence, with PAM SEQ ID NO: 114).

Following gene editing, on-target amplicon analysis was conducted aroundthe CD70 locus in TRAC−/β2M−/CD70−/anti-CD70 CAR+ cells (generated asdescribed in Example 3).

An initial PCR was performed using the KAPA HiFi PCR kit (KapaBiosystems, Wilmington, Mass.). 100 ng of input gDNA was combined with10 uM of each primer. The CD70_F and CD70_R primers were paired toamplify the CD70 locus (Table 7).

TABLE 7 Primers for CD70 amplicon library preparation CD70_FTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGcccaac ttttccatctcaactcaccccaagtg (SEQ ID NO: 127) CD70_R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGcccctcctgcgctagcgga (SEQ ID NO: 128)

Analysis of the CD70 locus in a population of T cells followingCRISPR/Cas9 gene editing to produce TRAC⁻/β2M⁻/anti-CD70 CAR+ T cellsresults in specific indel frequencies and edited gene sequences at theCD70 locus (Table 8; deletions as dashes and insertions in bold). Twocell populations of edited cells were generated from two different donorT cells (1 and 2). The populations of edited T cells from each donorwere analyzed in replicate: 1A/1B and 2A/2B.

TABLE 8 SEQ ID Std. NO: Gene Edited Sequence 1A 1B 2A 2B Mean Dev. 129CACACCACGAGGCAGATCACCAAGCCCGCG-- 10.4% 11.1% 14.4% 14.8% 12.7% 0.022CAATGGGACCAAAGCAGCCCGCAGGACG 130 CACACCACGAGGCAGATCACCAAGCCCGCGA  8.7%10.0% 11.3% 11.1% 10.3% 0.012 ACCAATGGGACCAAAGCAGCCCGCAGGACG 131CACACCACGAGGCAGATC------------  8.2%  7.8%  7.1%  6.8%  7.5% 0.006ACCAATGGGACCAAAGCAGCCCGCAGGACG 132 CACACCACGAGGCAGATCACCAAGCCCGCG-  3.9% 4.5%  4.2%  4.3%  4.2% 0.002 CCAATGGGACCAAAGCAGCCCGCAGGACG 133CACACCACGAGGCAGATCACCAAGCCCGC-  2.2%  2.5%  2.4%  2.6%  2.4% 0.002ACCAATGGGACCAAAGCAGCCCGCAGGACG 134CACACCACGAGGCAGATCACCA-------------------  2.9%  2.3%  2.0%  2.0%  2.3%0.004 ------AGCCCGCAGGACG

Example 2. Generation of T Cells with Multiple Gene Knockouts

This example describes the use of CRISPR/Cas9 gene editing technology toproduce human T cells that lack expression of two, three or four genessimultaneously. Specifically, the

T cell receptor (TCR) gene (gene edited in the TCR Alpha Constant (TRAC)region), the β2-microglobulin (β2M) gene, the Cluster of Differentiation70 (CD70) gene and/or the programmed cell death 1 (PD-1 or PD1) genewere edited by CRISPR/Cas9 gene editing to produce T cells deficient intwo or more of the listed genes. The following abbreviations are used inthe Figures for brevity and clarity:

2× KO: TRAC⁻/β2M⁻

3× KO (PD-1): TRAC⁻/β2M⁻/PD-1⁻

3× KO (CD70): TRAC⁻/β2M⁻/CD70⁻

4× KO: TRAC⁻/β2M⁻/PD-1⁻/CD70⁻

Activated primary human T cells were electroporated with Cas9:gRNA RNPcomplexes. The nucleofection mix contained the Nucleofector™ Solution,5×10⁶ cells, 1 μM Cas9, and 5 μM gRNA (as described in Hendel et al.,Nat Biotechnol. 2015; 33(9):985-989, PMID: 26121415). For the generationof double knockout T cells (2× KO), the cells were electroporated withtwo different RNP complexes, each containing Cas9 protein and one of thefollowing sgRNAs: TRAC (SEQ ID NO: 40) and β2M (SEQ ID NO: 41) at theconcentrations indicated above. For the generation of triple knockout Tcells (3× KO), the cells were electroporated with three different RNPcomplexes, each RNA complex containing Cas protein and one of thefollowing sgRNAs: (a) TRAC (SEQ ID NO: 40), β2M (SEQ ID NO: 41), andPD-1 (SEQ ID NO: 42) at the concentrations indicated above; or (b) TRAC(SEQ ID NO: 40), β2M (SEQ ID NO: 41), and CD70 (SEQ ID NO: 36 or 37) atthe concentrations indicated above. For the generation of quadrupleknockout T cells (4× KO), the cells were electroporated with fourdifferent RNP complexes, each RNA complex containing Cas9 protein andone the following sgRNAs: TRAC (SEQ ID NO: 40), β2M (SEQ ID NO: 41),PD-1 (SEQ ID NO: 42), and CD70 (SEQ ID NO: 36 or 37) at theconcentrations indicated above. The unmodified versions (or othermodified versions) of the gRNAs may also be used (e.g., SEQ ID NOS: 30,31, 32, 26, and/or 27). Sequences in Tables 5 and 9.

TABLE 9 gRNA Sequences/Target Sequences Name Unmodified SequenceModified Sequence TRAC sgRNA AGAGCAACAGUGCUGUGGCC A*G*A*GCAACAGUGCUGUGGguuuuagagcuagaaauagcaaguuaaaau CCguuuuagagcuagaaauagcaaguuaaaaggcuaguccguuaucaacuugaaaaagu aauaaggcuaguccguuaucaacuugaaaaggcaccgagucggugcUUUU aguggcaccgagucggugcU*U*U*U (SEQ ID NO: 30)(SEQ ID NO: 40) TRAC sgRNA AGAGCAACAGUGCUGUGGCC A*G*A*GCAACAGUGCUGUGGspacer (SEQ ID NO: 98) CC (SEQ ID NO: 108) β2M sgRNAGCUACUCUCUCUUUCUGGCC G*C*U*ACUCUCUCUUUCUGGguuuuagagcuagaaauagcaaguuaaaau CCguuuuagagcuagaaauagcaaguuaaaaggcuaguccguuaucaacuugaaaaagu aauaaggcuaguccguuaucaacuugaaaaggcaccgagucggugcUUUU aguggcaccgagucggugcU*U*U*U (SEQ ID NO: 31)(SEQ ID NO: 41) β2M sgRNA GCUACUCUCUCUUUCUGGCC G*C*U*ACUCUCUCUUUCUGGspacer (SEQ ID NO: 99) CC (SEQ ID NO: 109) PD-1 sgRNACUGCAGCUUCUCCAACACAU C*U*G*CAGCUUCUCCAACACguuuuagagcuagaaauagcaaguuaaaau AUguuuuagagcuagaaauagcaaguuaaaaggcuaguccguuaucaacuugaaaaagu aauaaggcuaguccguuaucaacuugaaaaggcaccgagucggugcUUUU (SEQ ID aguggcaccgagucggugcU*U*U*U NO: 32)(SEQ ID NO: 42) PD-1 sgRNA CUGCAGCUUCUCCAACACAU C*U*G*CAGCUUCUCCAACACspacer (SEQ ID NO: 100) AU (SEQ ID NO: 110)

About one (1) week post electroporation, cells were either leftuntreated or treated with phorbol myristate acetate (PMA)/ionomycinovernight. The next day cells were processed for flow cytometry (see,e.g., Kalaitzidis D et al. J Clin Invest 2017; 127(4): 1405-1413) toassess TRAC, β2M, PD-1, and CD70 expression levels at the cell surfaceof the edited cell population. The following primary antibodies wereused (Table 10):

TABLE 10 Antibodies Anti- body Clone Fluor Catalogue # Dilution For 1TCR BW242/412 PE 130-091-236 (Miltenyi) 1:100 1 μL β2M 2M2 PE-Cy7 316318(Biolegend) 1:100 1 μL PD-1 EH12.2H7 PE 329906 (Biolegend) 1:100 1 μLCD70 113-16 FITC 355105 (Biolegend) 1:100 1 μL

Tables 11 and 12 show highly efficient multiple gene editing. For thedouble-knock cells (2× KO; TRAC⁻/β2M⁻), 83% of viable cells lackedexpression of TCR and β2M (Table 11; 3× KO (PD1)). For the tripleknockout cells, 70% of viable cells lacked expression of TCR, β2M, andPD-1 (Table 11); and 80% of viable cells lacked expression of TCR, β2M,and CD70 irrespective of the CD70 gRNA used (Table 12). For thequadruple knockout cells (4× KO), 78% of viable cells lacked expressionof TCR, β2M, PD-1, and CD70 (FIG. 1).

TABLE 11 % of viable cells lacking expression in 2KO and 3KO (PD1) cellpopulations TRAC KO β2M KO PD1 KO 2 KO 3 KO (PD1) 2KO 98% 85% NA 83% NA3 KO (PD1) 98% 73% 99% NA 70%

TABLE 12 % of viable cells lacking expression in 3KO (CD70) cellpopulations TRAC KO β2M KO CD70 KO 3KO (CD70) 3KO (CD70) (T7) 99% 79%99% 80% 3KO (CD70) (T8) 99% 82% 99% 80%

To assess whether triple and quadruple gene editing in T cells affectscell expansion, cell numbers were enumerated among double, triple, andquadruple gene edited T cells (unedited T cells were used as a control)over a two week period of post editing. 5×10⁶ cells were generated andplated for each genotype of T cells.

As shown in FIG. 2, cell proliferation (expansion) continued over thepost-electroporation window test. Similar cell proliferation wasobserved among the double (β2M−/TRAC−), triple (β2M−/TRAC−/PD-1−, orβ2M−/TRAC−/CD70−), and quadruple (β2M−/TRAC−/PD-1−/CD70) knockout Tcells, as indicated by the number of viable cells. These data suggestthat multiple gene editing (up to triple and quadruple, with CD70 andPD-1 genes) does not impact T cell health as measured by T cellproliferation.

Example 3. Generation of CAR T Cells Lacking CD70 and/or PD1

Generation of Anti-CD70 CAR T Cells with Multiple Knockouts

This example describes the production of allogeneic human T cells thatlack expression of the TCR gene, β2M gene, CD70 gene and/or PD1 gene,and express a chimeric antigen receptor (CAR) targeting CD70. Thesecells are designated TCR⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ or 3× KO (CD70) CD70CAR⁺; TCR⁻/β2M⁻/PD1⁻/anti-CD70 CAR⁺ or 3× KO (PD1) CD70 CAR⁺;TCR⁻/β2M⁻/PD1⁻/CD70⁻/anti-CD70 CAR⁺ or 4× KO CD70 CAR⁺ in the Figures.

A recombinant adeno-associated adenoviral vector, serotype 6 (AAV6) (MOI50,000) comprising the nucleotide sequence of SEQ ID NO: 43 (comprisingthe donor template in SEQ ID NO: 44, encoding anti-CD70 CAR comprisingthe amino acid sequence of SEQ ID NO: 46) was delivered with Cas9:sgRNARNPs (1 μM Cas9, 5 μM gRNA) to activated allogeneic human T cells. Thefollowing sgRNAs were used: TRAC (SEQ ID NO: 40), β2M (SEQ ID NO: 41),CD70 (SEQ ID NO: 36 or 37) and PD1 (SEQ ID NO: 42). The unmodifiedversions (or other modified versions) of the gRNAs may also be used(e.g., SEQ ID NOS: 30, 31, 32, 26, and/or 27). About one (1) week postelectroporation, cells were processed for flow cytometry to assess TRAC,β2M, CD70, and PD1 expression levels at the cell surface of the editedcell population. The following primary antibodies were used (Table 13):

TABLE 13 Antibodies Antibody Clone Fluor Catalogue # Dilution TCRBW242/412 PE 130-091-236 (Miltenyi) 1:100 β2M 2M2 PE-Cy7 316318(Biolegend) 1:100 CD70 113-16 FITC 355105 (Biolegend) 1:100 PD-1EH12.2H7 PE 329906 (Biolegend) 1:100

T cell Proportion Assay. The proportions of CD4+ and CD8+ cells werethen assessed in the edited T cell populations by flow cytometry usingthe following antibodies (Table 14):

TABLE 14 Antibodies Antibody Clone Fluor Catalogue # Dilution CD4 RPA-T4BV510 300545 (Biolegend) 1:100 CD8 SK1 BV605 344741 (Biolegend) 1:100High efficiency gene editing and CAR expression was achieved in theedited anti-CD70 CAR T cell populations. In addition, editing did notadversely alter CD4/CD8 T cell populations. FIG. 3 shows highlyefficient gene editing and anti-CD70 CAR expression in the tripleknockout CAR T cell. More than 55% of viable cells lacked expression ofTCR, β2M, and CD70, and also expressed the anti-CD70 CAR. FIG. 4 showsthat normal proportions of CD4/CD8 T cell subsets were maintained in theTRAC−/β2M−/CD70−/anti-CD70 CAR+ cells, suggesting that these multiplegene edits do not affect T cell biology as measured by the proportion ofCD4/CD8 T cell subsets.

FIG. 5 shows show highly efficient gene editing and anti-CD70 CARexpression in the quadruple knockout CAR T cell. Greater than 60% ofviable cells lacked expression of TCR, β2M, PD-1, and CD70, andexpressed the anti-CD70 CAR. FIG. 6 shows that normal proportions ofCD4/CD8 T cell subsets were maintained in theTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ cells, suggesting that thesemultiple gene editing do not affect T cell biology as measured by theproportion of CD4+/CD8+ T cell subsets.

Generation of Anti-BCMA CAR T Cells with Multiple Knockouts

This example describes the production of allogeneic human T cells thatlack expression of the TCR gene, the β2M gene, the PD-1 gene, and/or theCD70 gene, and also express a chimeric antigen receptor (CAR) targetingB-cell maturation antigen (BCMA).

A recombinant adeno-associated adenoviral vector, serotype 6 (AAV6)comprising the nucleotide sequence of SEQ ID NO: 54 (comprising thedonor template in SEQ ID NO: 55, encoding anti-BCMA CAR comprising theamino acid sequence of SEQ ID NO: 57) was delivered with Cas9:gRNA RNPs(1 μM Cas9, and 5 μM gRNA) to activated allogeneic human T cells. Thefollowing gRNAs were used: TRAC (SEQ ID NO: 40), β2M (SEQ ID NO: 41),PD-1 (SEQ ID NO: 42), and CD70 (SEQ ID NO: 36 or 37). The unmodifiedversions (or other modified versions) of the gRNAs may also be used(e.g., SEQ ID NOS: 30, 31, 32, 26 and/or 27). About one (1) week postelectroporation, cells were processed for flow cytometry as describedabove for anti-CD70 CAR+ T cells, with the following difference.Anti-BCMA CAR expression was detected using biotinylated recombinanthuman BCMA (ACROS Cat# BC7-H82F0). The double and quadruple knockoutanti-BCMA CAR⁺ cells were then characterized as described herein.

FIGS. 7A-7B shows highly efficient gene editing of the TRAC gene, β2Mgene, the CD70 gene and the PD-1 gene. FIG. 7C shows high expression ofthe anti-BCMA CAR+ cells in double knockout and quadruple knockoutcells.

Generation of Anti-CD19 CAR T Cells with Multiple Knockouts

Allogeneic human T cells were generated that express a chimeric antigenreceptor (CAR) targeting CD19 and lack the expression of the TCR gene ,the ββ2M gene, and optionally the CD70 gene.

To generate the allogeneic T cells, activated primary human T cells wereelectroporated with Cas9:gRNA RNP complexes and infected withadeno-associated adenoviral vectors (AAVs) containing anti-CD19 CARdonor template with homology to the TRAC locus. Recombinant AAV serotype6 (AAV6) comprising the nucleotide sequence of SEQ ID NO: 155(comprising the donor template in SEQ ID NO: 156, encoding anti-CD19 CARcomprising the amino acid sequence of SEQ ID NO: 149) was delivered withCas9:sgRNA RNPs (1 μM Cas9, 5 μM gRNA) to activated human T cells. Thefollowing sgRNAs were used to knock-out the respective genes: TRAC (SEQID NO: 40), β2M (SEQ ID NO: 41), CD70 (SEQ ID NO: 36). The unmodifiedversions (or other modified versions) of the gRNAs may also be used(e.g., SEQ ID NOs: 30, 21 or 27). About one (1) week postelectroporation, cells were processed for flow cytometry as describedabove for anti-CD70 CAR+ T cells, with the following difference.Anti-CD19 CAR expression was detected using biotinylated recombinanthuman CD19 (ACROBIOSYSTEMS INC; CD9-H825). The CD70 deficient anti-CD19CAR⁺ T cells were then characterized as described herein.

Generation of anti-CD33 CAR T Cells with Multiple Knockouts

Allogeneic human T cells were generated that express a chimeric antigenreceptor (CAR) targeting CD33 and lack the expression of the T cellreceptor (TCR) gene (gene edited in the TCR Alpha Constant (TRAC)region), the β2-microglobulin (β2M) gene, and optionally the CD70 gene.

To generate the allogeneic T cells, activated primary human T cells wereelectroporated with Cas9:gRNA RNP complexes and infected withadeno-associated adenoviral vectors (AAVs) containing anti-CD33 CARdonor template with homology to the TRAC locus. Recombinant AAV serotype6 (AAV6) comprising the nucleotide sequence of SEQ ID NO: 87 (comprisingthe donor template in SEQ ID NO: 135, encoding anti-CD33 CAR comprisingthe amino acid sequence of SEQ ID NO: 139was delivered with Cas9:sgRNARNPs (1 μM Cas9, 5 μM gRNA) to activated human T cells. The followingsgRNAs were used to knock-out the respective genes: TRAC (SEQ ID NO:40), β2M (SEQ ID NO: 41), CD70 (SEQ ID NO: 36). The unmodified versions(or other modified versions) of the gRNAs may also be used (e.g., SEQ IDNOs: 30, 21 or 27).

Populations of TCR+ T cells (no RNP) and TRAC−/β2M-T cells (TCR and β2Mdeficient cells without a CAR) were similarly generated for use ascontrols. About one (1) week post electroporation, cells were processedfor flow cytometry as described above for anti-CD70 CAR+ T cells, withthe following difference. Anti-CD33 CAR expression was detected usingbiotinylated recombinant human CD33 (data not shown). The CD70 knockoutanti-CD33 CAR⁺ T cells were then characterized as described herein.

Characterization of CD4/CD8 Cell Populations in Anti-CD33 CAR T Cellswith CD70 Knock-Out

CD33 can be expressed on T cells with higher levels observed on culturedCD4 cells than CD8 cells. During the course of producing anti-CD33 CAR-Tcells CD4 cells become substantially reduced due to fratricide. As shownin FIG. 8, anti-CD33 CAR-T cell cultures with intact CD70 displayed a97% reduction in CD4+cells over a 3 week culture period, while culturesof cells with disrupted CD70 showed only a 61% reduction over this timecourse. Thus disrupting the CD70 gene appears to reduce the fratricideobserved in the anti-CD33 CAR-T cell cultures. Without wishing to bebound by theory, this effect may occur through an immune stimulatoryfunction which could be potentiated by CD70/CD27 interactions, andgenetic disruption of CD70 results in more balanced CD4/CD8 ratios thatmay be more optimal for therapeutic benefit in malignancy.

Example 4: CD70 KO Improves Cell Proliferation Effect of CD70 KO on CellProliferation of Anti-CD33 CAR T Cells In Vitro

To assess the ability of cells to expand in cytokine containing media(IL-2+IL-7), anti-CD33 CAR T cells were utilized. Specifically, 5×10⁶total anti-CD33 CAR T cells comprising a double knockout (TRAC−/B2M−) ortriple knockout (TRAC−/B2M−/CD70−) were generated as described inExample 3, plated and allowed to grow in a 10 mL volume of cytokinecontaining media. After 1 week cells were counted. 5×10⁶ cells from theprevious culture were then replated in 10 mL volume (fresh cytokinecontaining media) and 1 week later the total number of cells wereenumerated. Allogeneic anti-CD33 CAR-T cells containing a disruption inthe CD70 gene expanded to greater levels on the first and second week ofreplating (FIG. 9). These data show that CD70 knockout can result ingreater cell yields in culture.

Effect of CD70 KO on Cell Proliferation of Anti-CD19 CAR T Cells InVitro

To further assess the ability of cells to expand in cytokine containingmedia (IL-2+IL-7), anti-CD19 CAR T cells were utilized. Specifically,5×10⁶ total anti-CD19 CAR T cells comprising a double knockout(TRAC−/B2M−) or triple knockout (TRAC−/B2M−/CD70−) were generated asdescribed in Example 3, plated and allowed to grow in a 10 mL volume ofcytokine containing media. After 1 week cells were counted. 5×10⁶ cellsfrom the previous culture were then replated in 10 mL volume (freshcytokine containing media) and 1 week later the total number of cellswere enumerated. Allogeneic anti-CD19 CAR-T cells containing adisruption in the CD70 gene expanded to greater levels on the first andsecond week of replating as compared to control cells without a CD70gene distruption (FIG. 10). These data show that CD70 knockout canresult in greater cell yields in culture.

Effect of CD70 KO on Cytokine Driven Proliferation and Apoptosis ofAnti-BCMA CAR T Cells In Vitro

Cytokine driven proliferation. To evaluate the effect of CD70 and/or PD1knockout on cell proliferation, anti-BCMA CAR T cells were utilized.Anti-BCMA CAR T cells were generated as described in Example 3. Thefollowing groups of edited T cells were generated:

TRAC−/B2M−/anti-BCMA CAR+ (Control; 2KO, BCMA CAR+)

TRAC−/B2M−/CD70−/anti-BCMA CAR+ (3KO (CD70), BCMA CAR+)

TRAC−/β2M−/PD1−/anti-BCMA CAR+ (3KO (PD1), BCMA CAR+)

TRAC−/β2M−/CD70−/PD1−/anti-BCMA CAR+ (4KO, BCMA CAR+)

Edited cells were enriched for TRAC−/B2M− cells by magnetic depletion ofCD3+B2M+cells. Briefly, cells were labelled with anti-CD3 Biotin(Biolegend Cat#300404) anti-β2M Biotin (Biolegend Cat #316308)antibodies, each at 0.5 μg per 1 ×10⁶ cells in 100 μl volume at 4° C.for 15 min, washed and incubated with Streptavidin labelled magneticmicrobeads (Miltenyi Biotech, 130-048-101) for 15min at 4° C. Cells wereresuspended in buffer and passed through LS columns (Miltenyi Biotech,130-042-401) according to the manufacturer's protocol. To determine theeffect of CD70 or PD1 on IL-2/IL-7 driven T cell proliferation, theedited T cells (1E6 cells/ml) were cultured in growth medium (X-vivomedium (04-744, Lonza), supplemented with 5% human AB serum (HP1022,Valley Biomedical)), 50 ng/ml IL-2 (rhlL-2; 130-097-745, MiltenyiBiotech) and 10 ng/ml IL-7 (rhlL-7; Cellgenix 001410-050) for up to fourweeks. At indicated days, the cells were counted and re-seeded in freshmedium at 1.5E6 cells/ml in appropriate culture dishes.

FIGS. 11 and 12 show that knockout of CD70 improved IL-2/IL-7 drivenproliferation of anti-BCMA CAR T cells in vitro, as compared to CD70sufficient controls (i.e. anti-BCMA CAR T cells comprising endogenousCD70). FIG. 11 also shows the CD70 KO can improve health andproliferation competence of anti-BCMA CAR T cells even when the T cellsfrom this donor appear to be in significant decline after 17 days whenthe CD70 gene is intact (as shown by the reduced cell numbers of donor1(FIG. 11) compared to donor 2 (FIG. 12)). This property of maintainingT cell health (enabled by KO of the CD70 gene) is broadly applicable tomany aspects of CAR T development including: extended expansion duringmanufacturing increasing yield and consistency, rescue ofexhausted/unhealthy T cells enabling potentially lower doses in patientsand more robust responses, combination with other KOs that may be moredetrimental to T cell health but have other advantages such asovercoming suppression of T cell activity (e.g. PD1 KO). As shown inFIGS. 11 and 12, deleting the PD1 gene by itself shows no benefit to CART cell expansion but when combined with a CD70 KO shows synergisticeffects.

Apoptosis. The effect of CD70 KO on apoptotic cell death of anti-BCMACAR+ T cells following exposure to antigen was evaluated in an antigenrechallenge assay. Briefly, to achieve antigen exposure, anti-BCMA CAR+T cells were exposed to plate-adhered recombinant BCMA protein. Plateswith adhered antigen were prepared by coating 24 well plates withrecombinant BCMA protein in 1× PBS (1 μg/ml; biotinylated Human BCMAProtein, ACRO Biosystems) overnight at 4° C. and then washing awayunbound antigen. Following the wash, antigen-bound plates were then usedto challenge anti-BCMA CAR+ T cells either with or without a CD70knockout. The 2× KO (TRAC−/B2M−) anti-BCMA CAR+ T cells and 3× KO(TRAC−/B2M−/CD70−) anti-BCMA CAR+ T cells (1×10⁶ cells/ml) were exposedto plate-bound recombinant BCMA protein (1 μg/ml) for 24 hours in growthmedium (X-vivo medium (04-744, Lonza), 5% human AB serum (HP1022, ValleyBiomedical)) supplemented with IL-2 (rhlL-2; 130-097-745, MiltenyiBiotech). Cells were then washed, counted and re-challenged (1×10⁶cells/ml) with fresh plate-bound antigen every 24 hours for a total ofthree consecutive re-challenges (24 hr, 48 hr, and 72 hr). At the end ofeach re-challenge, an aliquot of cells was washed and stained withfluorochrome-conjugated annexin V along with propidium iodide in annexinV binding buffer (BioLegend) for 15 minutes at room temperature. Cellswere then washed and resuspend in annexin V binding buffer for analysisby flow cytometry. The cells were counted at each time point and thecell count per ml was derived. For the calculation of fold-expansion ateach time point, the initial fold-expansion at time 0 was set at 1.Fold-expansion for all other time points were calculated by multiplyingthe cell count per ml at each time point by the fold-expansion per mlfor the prior time point. For example, the fold expansion at 72 hr wascalculated by multiplying the cell count per ml at 72 hr by the foldexpansion per ml at 48 hr.

FIG. 13 demonstrates that the deletion of CD70 (CD70 KO) rescuesanti-BCMA CAR+ T cells from apoptosis, as shown by the decrease in thepercentage of apoptotic cells following the second (48 hr) and third (72hr) rechallenge. Furthermore, the absence of CD70 expression inanti-BCMA CAR+ T cells surprisingly enhances the expansion of theanti-BCMA CAR+ T cells in response to antigen exposure (FIG. 14).

Effect of CD70 KO on Cell Proliferation of Anti-CD70 CAR T Cells InVitro

To further assess the impact of disrupting the CD70 gene in CAR T cells,anti-CD70 CAR T cells were generated as described in Example 3.Specifically, 3× KO (TRAC−/β2M−/CD70−) anti-CD70 CAR T cells weregenerated using two different gRNAs (T7 (SEQ ID NO: 36 and T8 (SEQ IDNO: 37)). After electroporation, cell expansion was assessed asdescribed in Example 2 by counting viable cells. FIG. 15 shows thattriple knockout TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells generated witheither T7 or T8 gRNAs exhibited greater cell expansion relative todouble knockout TRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cells. These data suggestthat knocking-out the CD70 gene gives a cell proliferation advantage toanti-CD70 CAR+ T cells.

Cell expansion was also assessed in the quadruple knockout,TRAC⁻/β2M⁻/PD-1/CD70⁻/anti-CD70 CAR⁺ T cells. These cells exhibitedgreater expansion relative to triple knockout TRAC⁻/β2M⁻/PD-1⁻/anti-CD70CAR⁺ T cells and to double knockout TRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cells(FIG. 16).

Example 5. CD70 KO Increases Durability and Potency of CAR T Cells InVitro

Cell Killing Function of Anti-CD19 CAR T Cells with CD70 Knock-Out

Following preparation of edited anti-CD19 CAR T cells as described inExample 3, the functional activity of the CAR T cells was verified usinga flow cytometry-based cytotoxicity assay. The anti-CD19 CAR T cells(TRAC−/β2M−/CD19 CAR+ and TRAC−/β2M−/CD70−/CD19 CAR+) were co-culturedwith one of two CD19-expressing cancer cell lines (target cells): Nalm6(ATCC cr13273) or Raji (ATCC cc1-86). The target cells were labeled with5 μM efluor670 (eBiosciences), washed and incubated in co-cultures withthe TRAC−/β2M−/anti-CD19 CAR+, or TRAC−/β2M−/CD70−/anti-CD19 CAR+ atvarying ratios (0.01, 0.05, 0.1, 0.5, 1:1 T cells:target cells). Thetarget cells were seeded at 50,000 cells per well in a 96-well, U-bottomplate. The co-culture was incubated overnight. After incubation, wellswere washed and media was replaced with 200 μL of media containing a1:500 dilution of 5 mg/mL DAPI (Molecular Probes). 25 μL of CountBrightbeads (Life Technologies) were then added to each well and the cellcultures were analyzed for cell viability by flow cytometry (i.e.,viable cells being negative for DAPI staining).

Percent cell lysis of the target cells (e.g.: Nalm6 or Raji cells) wasthen determined using the following formula:

Percent cell lysis=(1−((total number of target cells in a testsample)÷(total number of target cells in a control sample))×100;

wherein a test sample was target cells (e.g.: Nalm6 or Raji cells)co-cultured with 1) TRAC−/β2M−/CD19 CAR+ T cells or 2)TRAC−/β2M−/CD70−/CD19 CAR+ T cells; and

a control sample was target cells alone that had not been co-cultured.

Disruption of the CD70 gene led to enhanced cytolytic activity of theanti-CD19 CAR-T cells against the Raji cell line at low CAR-T to targetratios (FIG. 17, bottom panel). Disruption of CD70 did not enhanceanti-CD19 CAR-T activity against the Nalm6 cell line (FIG. 17, toppanel). Of note, the Nalm6 cell line is relatively easier to lyse bythese CAR-T cells (>80% lyses at 0.1:1 CAR-T cell to target ratio forNalm6 vs 0% for Raji at this ratio for the wild-type cells) likelyexplaining the lack of resulting increased efficacy due to CD70disruption in this assay against Nalm6 cells. The increased activityconferred by CD70 loss against the Raji cell line indicates that inchallenging tumor environments, particularly when CAR-T to tumor ratiosare low, CD70 loss may have substantial benefit to the CAR-T cells ineradicating tumor cells.

Cell Killing Function of Anti-CD33 CAR T Cells with CD70 Knock-Out

Following preparation of the edited anti-CD33 CAR+ T cells as describedin Example 3, the functional activity of the CAR T cells was verifiedusing a flow cytometry-based cytotoxicity assay. The anti-CD33 CAR Tcells (TRAC−/β2M−/CD33 CAR+ and TRAC−/β2M−/CD70−/CD33 CAR+) or control Tcells (no RNP) were co-cultured with the CD33-expressing cancer cellline MV4-11 (ATCC CRL-9591). The target cells were labeled with 5 μMefluor670 (eBiosciences), washed and incubated in co-cultures with theTRAC−/B2M−/anti-CD33 CAR+, TRAC−/β2M−/CD70−/anti-CD33 CAR+, or controlsat varying ratios (0.01:1, 0.03:1, 0.06:1, 0.125:1, 0.25:1, 0.5:1, or1:1 T cells:target cells). The target cells were seeded at 50,000 cellsper well in a 96-well, U-bottom plate. The co-culture was incubatedovernight. After 48 hrs, wells were washed and media was replaced with200 μL, of media containing a 1:500 dilution of 5 mg/mL DAPI (MolecularProbes). 25 μL of CountBright beads (Life Technologies) were then addedto each well and the cell cultures were analyzed for cell viability byflow cytometry (i.e., viable cells being negative for DAPI staining).

Percent cell lysis of the target cells (e.g.: MV4-11) was thendetermined using the following formula:

Percent cell lysis=(1−((total number of target cells in a testsample)÷(total number of target cells in a control sample))×100;

wherein a test sample was target cells (e.g.: MV4-11 cells) co-culturedwith 1) TRAC−/β2M−/CD33 CAR+ T cells; or 2) TRAC−/β2M−/CD70−/CD33 CAR+ Tcells, and

a control sample was target cells alone that had not been co-cultured.

Although both populations of anti-CD33 CAR T cells effectively killedMV4-11 cells, reaching nearly 100% cells kill at ratios of 0.5 CAR Tcell: MV4-11 cell, the TRAC−/B2M−/CD70−/CD33 CAR+ T cells demonstratedhigher cell killing at lower CAR T to cancer cell rations (FIG. 18).These data demonstrate that allogeneic anti-CD33 CAR T cells with theadditional CD70 knock-out are more efficacious at lower CAR T cell totarget cell ratios.

Cell Killing Function of Anti-CD70 CAR T Cells with CD70 Knock-Out

A cell killing assay was used to assess the ability of theTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ cells andTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ cells to kill a CD70⁺ adherentrenal cell carcinoma (RCC)-derived cell line (A498 cells). Adherentcells were seeded in 96-well plates at 50,000 cells per well and leftovernight at 37° C. The next day edited anti-CD70 CAR T cells were addedto the wells containing target cells at the indicated ratios. After theindicated incubation period, CAR T cells were removed from the cultureby aspiration and 100 μL Cell titer-Glo (Promega) was added to each wellof the plate to assess the number of remaining viable cells. The amountof light emitted per well was then quantified using a plate reader. Thecells exhibited potent cell killing of RCC-derived cells following24-hour co-incubation (FIG. 19). The anti-CD70 CAR T cells demonstratedhigher potency when CD70 was knocked out, which is clearly visible atlow T cell: A498 ratios (1:1 and 0.5:1) where cell lysis remains above90% for TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺, while cells lysis drops below90% for the TRAC⁻/β2M⁻/anti-CD70 CAR⁺. This suggests that knocking-outthe CD70 gene gives a higher cell kill potency to anti-CD70 CAR+ Tcells.

Example 6. Rechallenge of CD70 Deficient CAR T Cells In Vitro CD70Knockout Improves Anti-CD33 CAR+ T Cell Killing Upon Serial Rechallenge

To assess the ability of cells to expand after challenge and rechallengewith antigen-expressing cells (e.g.: target cells) anti-CD33 CAR T cellswere generated as described in Example 3 and utilized. Specifically,5×10⁶ total T cells were plated in the presence of 5×10⁶ irradiatedtarget cells (MV-4-11) and allowed to grow in a 10 mL volume. After 1week, cells were counted, 5×10⁶ cells from the previous culture werethen replated in 10 mL volume along with a fresh aliquot of 5×10⁶irradiated target cells and 1 week later the total number of cells wereenumerated. The process was repeated as indicated, each rechallengestarted with 5×10⁶ cells. The number of viable cells were counted asdescribed in Example 2. Allogeneic anti-CD33 CAR-T cells containing adisruption in the CD70 gene expanded to greater levels on the secondweek of after 2 challenges with MV-4-11 cells (FIG. 20). These data showthat CD70 can limit T-cell expansion in the presence of antigenexpressing cells and its loss can result in greater cell expansion afterantigen stimulation.

CD70 Knockout Improves Anti-CD19 CAR+ T Cell Killing Upon SerialRechallenge

To further assess the ability of cells to expand after challenge andrechallenge with antigen-expressing cells (e.g.: target cells) anti-CD19CAR T cells were generated as described in Example 3 and utilized.Specifically, 5×10⁶ total T cells were plated in the presence of 5×10⁶irradiated target cells (Nalm6) and allowed to grow in a 10 mL volume.After 1 week, cells were counted, 5×10⁶ cells from the previous culturewere then replated in 10 mL volume along with a fresh aliquot of 5×10⁶irradiated target cells and 1 week later the total number of cells wereenumerated. The process was repeated as indicated, each rechallengestarted with 5×10⁶ cells. The number of viable cells were counted asdescribed in Example 2. Allogeneic anti-CD19 CAR-T cells containing adisruption in the CD70 gene expanded to similar amounts during the first2 challenges. However, at three challenges allogeneic anti-CD19 CAR-Tcells containing a disruption in the CD70 gene expanded to greater levelon the third challenge with Nalm6 cells (FIG. 21). These data show thatthe presence of CD70 can limit T-cell expansion in the presence ofantigen expressing cells and its loss can result in greater cellexpansion after repeated antigen stimulation.

Knockout of CD70, or PD-1 plus CD70, Maintain Anti-CD70 CAR⁺ T CellKilling Upon Serial Rechallenge

The anti-CD70 CAR+ T cells generated above were serially rechallengedwith CD70+ kidney cancer cell line, A498, and evaluated for theirability to kill the CD70+ kidney cancer cell lines A498 or ACHN.

A498 cells were plated in a T25 flask and mixed at a ratio of 2:1(T-cell to A498) with 10×10⁶ anti-CD70 CAR⁺ T cells containing eithertwo (TRAC⁻/β2M⁻), three (TRAC⁻/β2M⁻/PD-1⁻) or (TRAC⁻/β2M⁻/CD70⁻)), orfour (TRAC⁻/β2M⁻/PD-1⁻/CD70⁻) gRNA edits.

Two or three days after each challenge, cells were counted, washed,resuspended in fresh T cell media, and re-challenged the next day withthe same ratio of two anti-CD70 CAR⁺ T cell per one A498 cell (2:1, CAR⁺T:target). Challenging of anti-CD70 CAR⁺ T cells with CD70+ A498 cellswas repeated 13 times. Three to four days following each exposure toA498 cells (and prior to the next rechallenge), aliquots of the culturewere taken and analyzed for the ability of the CAR T Cells to kill A498or ACHN target cells at a ratio of 2:1 (CAR T cell: Target cell). Cellkill was measured using Cell titer-glo (Promega). Prior to the firstchallenge with A498, anti-CD70 CAR+ T cells with 2× KO (TRAC⁻/β2M⁻), 3×KO (TRAC⁻/β2M⁻/CD70), 3× KO (TRAC⁻/β2M⁻/PD-1⁻), and 4× KO(TRAC⁻/β2M⁻/PD-1⁻/CD70⁻) each exhibited a target cell killing of A498cells approaching 100%. By challenge nine however, the 2× KO(TRAC⁻/β2M⁻) and 3× KO (TRAC⁻/β2M⁻/PD-1⁻) anti-CD70 CAR+ T cells inducedtarget cell killing of A498 cells below 40%, while 3× KO(TRAC⁻/β2M⁻/CD70) and 4× KO (TRAC⁻/β2M⁻/PD-1⁻/CD70⁻) anti-CD70 CAR⁺ Tcells exhibited target cell killing above 60% (FIG. 22A). The targetcell killing for 3× KO (TRAC⁻/β2M⁻/CD70) and 4× KO(TRAC⁻/β2M⁻/PD-1⁻/CD70⁻) anti-CD70 CAR⁺ T cells remained above 60% evenfollowing 13 re-challenges with A498 cells, demonstrating that theseCAR+ T cells were resistant to exhaustion.

Anti-CD70 CAR T cells were also evaluated for their ability to kill ACHNcells at a ratio of 2:1 (T-cell to ACHN) following serial rechallengewith A498 renal carcinoma cells (FIG. 22B). Prior to the first challengewith A498, the double knockout TRAC⁻/β2M⁻anti-CD70 CAR⁺ T cells, thetriple knockout TRAC⁻/β2M⁻/PD-1⁻anti-CD70 CAR⁺ T, the triple knockoutTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells and the quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T exhibited a cell kill efficiencyabove 62%, 47%, 73% and 81%, respectively.

After challenge five, the triple knockout TRAC⁻/β2M⁻/CD70⁻/anti-CD70CAR⁺ T cells and the quadruple knockout TRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70CAR⁺ T cells still efficiently killed above 55% of ACHN cells at a ratioof 2:1 (T-cell to ACHN), while the double knockout TRAC⁻/β2M⁻/anti-CD70CAR⁺ T cells and the triple knockout TRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ Tcell kill dropped below 11% of ACHN cells. This trend continued, whereinthe double knockout TRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cells and the tripleknockout TRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ T cells failed to survivebeyond 10 rechallenges. In contrast, the triple knockoutTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells and the quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells continued to expand inculture and to kill greater than 30% of ACHN cells at a ratio of 2:1(T-cell to ACHN) following two rechallenges.

The data demonstrate that the 4× KO, CD70 CAR⁺ T cells and the 3× KO(CD70), CD70 CAR⁺ cells are more potent than the 2× KO, CD70 CAR⁺ T or3× KO (PD1), CD70 CAR+ T cells. In addition, the 3× (CD70) KO and 4× KOprevents T cell exhaustion.

After 5 rechallenges the cells were evaluated for their ability to killcancer cells. Surprisingly, the 3KO and 4KO anti-CD70 CAR+ T cellsremained highly effective at killing cancer cells (FIG. 23A) even aftermultiple cancer cell challenges. The cell killing effect of theanti-CD70 CAR+ T cells on ACHN cells is reproducible at even at reducedeffector to target cell ratios of 1:1, 0.5:1, and 0.25:1. (FIG. 23A).

To ensure long-term benefit upon CAR T treatment, CAR T cells should beable to identify and eradicate their target cells over a long period oftime, to rule out the possibility of cancer cell escape from CAR-Tmediated cell kill. The in vitro re-challenge assay mimics a recurrentencounter of CAR-T cells with target cells over several cycles of CAR-Tcell activation. These data demonstrate the superiority of the tripleknockout TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells and of the quadrupleknockout TRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells, in sustainingmultiple challenges with kidney cancer cells, without showing reductionof their target cell killing ability, as compared to the double knockoutTRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cells and the triple knockoutTRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ T cells.

Exhaustion and activation markers were also measured by flow cytometryin the anti-CD70 CAR+ T cells following rechallenge. After 8 challenges,the Triple (TRAC−/β2M−/CD70−) and Quadruple (TRAC−/β2M−/PD1−/CD70−) KOanti-CD70 CAR+ T cells exhibited higher activation marker LAG3expression than the Double (TRAC−/β2M−) and Quadruple(TRAC−/β2M−/PD1−/CD70−) KO anti-CD70 CAR+ T cells, consistent with theirlevel of high cell kill activity. It was observed that PD1 expressionwas lower in the Triple (TRAC−/β2M−/CD70−) anti-CD70 CAR+T cells(similar to Triple (TRAC−/β2M−/PD1−) and Quadruple(TRAC−/β2M−/PD1−/CD70−) KO anti-CD70 CAR+T cells) compared to the Double(TRAC−/β2M−) anti-CD70 CAR+T cells, suggesting that knocking-out CD70has an effect on the downregulation of the exhaustion marker PD1expression in the Anti-CD70 CAR+T cells. (FIG. 23B).

Knockout of PD-1 and CD70 Maintains Anti-BCMA CAR⁺ T Cell Killing UponSerial Rechallenge

The anti-BCMA CAR+ T cells generated as described in Example 3 wereserially rechallenged with and evaluated for their ability to kill theBCMA+ multiple myeloma cell line MM.1S (ATCC CRL-2974). The ability tosecrete cytokines upon serial T cell activation through CAR engagementwas also measured after each rechallenge. MM.1S cells were labeled with5 μM eFlour670 and mixed at a ratio of 2:1 (MM.1S to T-cell) in a 6 welltissue culture dish with 1×10⁶ anti-BCMA CAR⁺ T cells containing eithertwo (TRAC⁻/β2M⁻) or four ((TRAC⁻/β2M⁻/PD-1⁻/CD70⁻) gRNA edits. One dayfollowing exposure to MM.1S cells, an aliquot of the culture was takenand analyzed for both target cell kill & IFN-g secretion by CAR-T cells.To measure cytokine release, T cells and target cells were co-incubatedfor 24 hours at the ratios indicated. Supernatant media was collectedfor use in IL-2 or IFNγ ELISAs (RD Systems) on a new plate following themanufacturer's instructions (RD Systems). To quantify cell killing,cells were washed, media was replaced with 200 mL of media containing a1:500 dilution of 5 mg/mL DAPI (Molecular Probes) (to enumeratedead/dying cells). Finally, 25 mL of CountBright beads (LifeTechnologies) was added to each well. Cells were then processed by flowcytometry.

-   -   1) Cells/mL=((number of live target cell events)/(number of bead        events))×((Assigned bead count of lot (beads/50 μL))/(volume of        sample))    -   2) Total target cells were calculated by multiplying        cells/mL×the total volume of cells.    -   3) The percent cell lysis was then calculated with the following        equation:

% Cell lysis=(1−((Total Number of Target Cells in Test Sample)/(TotalNumber of Target Cells in Control Sample))×100

Two or three days after each challenge, cells were counted, washed,resuspended in fresh T cell media, and rechallenged with the same ratioof one anti-BCMA CAR⁺ T cell per two eFlour670 labeled MM.15 cells.Challenging of anti-BCMA CAR⁺ T cells with BCMA+ MM.1S cells wasrepeated 10 sequential times. Prior to any challenge with MM.1S cells,co-incubation of either 2× KO (TRAC−/B2M−) or 4× KO(TRAC−/B2M−/CD70−/PD-1−) anti-BCMA CAR+ T cells with MM.1S cellsresulted in complete killing of target cells. Additionally, IFNγproduction by both 2× KO and 4× KO anti-BCMA CAR+ T cells was similar.Following a 4th rechallenge with MM.1S cells however, target cellkilling and IFNγ production by 2× KO anti-BCMA CAR+ T cells decreasedrelative to that induced by 4× KO anti-BCMA CAR+ T cells. By the 8threchallenge, target cell killing was only approximately 20% for 2× KOanti-BCMA CAR+ T cells, while both IFNg and target cell killing by 4× KOanti-BCMA CAR+ T cells remained comparable to that seen prior to anychallenge with MM.1S cells (FIGS. 24A-24B). In addition, the quadrupleknockout anti-BCMA CAR T cells showed higher proliferation in responseto exposure to target cells (FIG. 24C).

To ensure long-term benefit upon CAR T treatment, CAR T cells should beable to identify and eradicate their target cells over long period oftime, to rule out the possibility of cancer cell escape from CAR-Tmediated cell killing. The in vitro rechallenge assay mimics a recurrentencounter of CAR-T cells with target cells over several cycles of CAR-Tcell activation, therefore demonstrating the superiority of the 4×knockout TRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-BCMA CAR⁺ T cells, in sustainingmultiple challenges with target cells, without showing reduction of cellkilling ability, as compared to the double knockout TRAC⁻/β2M⁻/anti-BCMACAR+ T cells (FIGS. 24A-24C).

Example 7. CD70 KO Overcomes Challenge of Excess Inhibitory Molecules

Comparison of the Effects of Multi-Knockout Anti-CD70 CAR+ T cells onA498-PD-L1 Renal Carcinoma Cells

Cell Kill Assay. The ability of multi-gene edited anti-CD70 CAR⁺ cellsto kill A498 renal carcinoma cells overexpressing PD-L1 was determinedusing the cell kill assay described herein. To create cellsoverexpressing PD-L1 (CD274), A498 cells were infected with lentivirusencoding a PD-L1 cDNA and a puromycin resistance gene (Genecopoeia).After selection with puromycin, cells were stained with an anti-PD-L1antibody to assess expression of PD-L1. The A498 cells expressing PD-L1are referred to as A498-PD-L1 and were used in the functional assaysdescribed.

The TRAC⁻/β2M⁻/anti-CD70 CAR⁺ (2× KO, CD70 CAR⁺),TRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ (3× KO (PD-1), CD70 CAR⁺),TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ (3× KO (CD70), CD70 CAR⁺) andTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ (4× KO, CD70 CAR⁺) T cells wereincubated with the A498-PD-L1 cells at a CAR T cell:A498-PD-L1 targetcells ratio of 2:1 (FIG. 25A), 1:1 (FIG. 25B), or 0.5:1 (FIG. 25C). TheCD70 knockout cells exhibited potent cell killing of RCC-derived cellsfollowing 24-hour co-incubation (FIGS. 25A-25C). The cells with PD1knockout alone did not effectively lyse cells in the presence of PD-L1overexpression. However, the CD70 knockout was able to rescue the PD1knockout and enhanced cell lysis was observed in the CART cells withCD70 KO and PD1 KO. These data demonstrate that the loss of CD70 on thesurface of these CAR-T cells enhances their function even in thepresence of highly immune suppressive molecules expressed by tumor cellssuch as PD-L1.

Cytokine Release Assay. A cytokine release assay was performed asdescribed herein. The ability of the double knockout, triple knockout,and quadruple knockout anti-CD70 CAR⁺ T cells to produce IL-2 and IFN-gwhen co-cultured in the presence of A948-PD-L1 cells following 24-hourco-incubation at a ratio (CAR T cell:A948-PD-L1 target cell) of 1:1 wasassessed using an ELISA assay. IL-2 and IFN-g from supernatants of cellco-cultures were measured. The quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells secreted the highestlevels of IFN-g (FIG. 26A) and IL-2 (FIG. 26B) when cultured withA948-PD-L1 cells. These data demonstrate the knock-out of CD70 enhancesCAR-T cells secretion of cytokines even in the presence of highly immunesuppressive molecules expressed by tumor cells such as PD-L1. Theknockout of CD70 together with a knockout of PD-1 in CAR-T cells furtherenhances the effect. Without wishing to be bound by theory, it isbelieved knocking-out CD70 in anti-CD70 CAR+ T cells can rescue thedetrimental phenotypes of other cell knockouts (e.g.: PD1). These datademonstrate that knocking-out CD70 in CAR T cells enhances target cellkilling and CAR T cell function in a highly immune suppressive context.

Example 8. CD70 Knockout Improves In Vivo Efficacy

Efficacy of CD70 and PD1 knockout in Anti-CD70 CART cells: theSubcutaneous Renal Cell Carcinoma Tumor Xenograft Model in NOG Mice

Treatment in Small Tumor Model

The ability of T cells expressing a CD70 CAR to eliminate kidneycarcinoma cells that express high levels of CD70 was evaluated in invivo using a subcutaneous renal cell carcinoma (A498) tumor xenograftmodel in mice.

CRISPR/Cas9 and AAV6 were used as above (see for example, Example 3) tocreate human T cells that lack expression of the TCR, β2M, CD70 and/orPD1 with concomitant expression from the TRAC locus using a CARconstruct targeting CD70 (SEQ ID NO: 45; SEQ ID NO: 46). In this exampleactivated T cells were first electroporated with 2, 3 or 4 distinctCas9:sgRNA RNP complexes containing sgRNAs targeting TRAC (SEQ ID NO:40), β2M (SEQ ID NO: 41), PD1 (SEQ ID NO: 42), and CD70 (SEQ ID NO: 36or 37). The DNA double stranded break at the TRAC locus was repaired byhomology directed repair with an AAV6-delivered DNA template comprisinga donor template (SEQ ID NO: 44; SEQ ID NO: 45) (encoding anti-CD70 CARcomprising the amino acid sequence of SEQ ID NO: 45) containing rightand left homology arms to the TRAC locus flanking a chimeric antigenreceptor cassette (−/+ regulatory elements for gene expression).

The resulting modified T cells are 2× KO (TRAC−/β2M−), 3× KO(TRAC−/β2M−/PD1− or TRAC−/β2M−/CD70−) and 4× KO (TRAC−/β2M−/PD1−/CD70−)anti-CD70 CAR+ (with 41BB costimulatory domain) T cells. The ability ofthese anti-CD70 CAR+ T cells to ameliorate disease caused by a CD70+renal carcinoma cell line was evaluated in NOG mice using methodsemployed by Translational Drug Development, LLC (Scottsdale, Ariz.). Inbrief, 12, 5-8 week old female, CIEA NOG(NOD.Cg-Prkdc^(scid)I12rg^(tm1Sug)/JicTac) mice were individually housedin ventilated microisolator cages, maintained under pathogen-freeconditions, 5-7 days prior to the start of the study. Mice received asubcutaneous inoculation of 5×10⁶ A498 renal carcinoma cells/mouse inthe right hind flank. When mean tumor size reached 25-75 mm³ (target of˜50 mm³), the mice were further divided into 5 treatment groups as shownin Table 15. On Day 1, treatment group 2 to 5 received a single 200 μlintravenous dose of anti-CD70 CAR+ T cells according to Table 15.

TABLE 15 Treatment groups T cell treatment Group CAR-T A498 cells (i.v.)N 1 None 5 × 10⁶ None 5 cells/mouse 2 2X KO, anti-CD70 5 × 10⁶ 1 × 10⁷ 5CAR+ T cells cells/mouse cells/mouse 3 3X KO (PD1), anti- 5 × 10⁶ 1 ×10⁷ 5 CD70 CAR+ T cells cells/mouse cells/mouse 4 3X KO (CD70,) anti- 5× 10⁶ 1 × 10⁷ 5 CD70 CAR+ T cells cells/mouse cells/mouse 5 4X KO (CD70,PD1), 5 × 10⁶ 1 × 10⁷ 5 anti-CD70 CAR+ T cells cells/mouse cells/mouse

Tumor volume was measured 2 times weekly from day of treatmentinitiation. By day 5 treatment with all four types of anti-CD70 CAR Tcells began to show a decrease in tumor volume and by day 22, all fourtypes of anti-CD70 CAR T cells completely eliminated CD70+ kidney cancertumors during the duration of the study until day 91 (FIG. 27A). Thesedata demonstrate that all four anti-CD70 CAR T cells can regress CD70+kidney cancer tumors in vivo.

To test the activity of the anti-CD70 CAR T cells after rechallenge,surviving mice were inoculated in the subcutaneous left hind flank with5×10⁶ A498 renal carcinoma cells/mouse on day 25. (Table 16). Sustainedefficacy was evaluated from day 46 onward. Results are shown in FIG.27B. At day 56, 5 out of 5 mice treated with 2×, CD70 CAR+ T cellsexhibited tumors regrowth post rechallenge, 4 out of 5 mice treated with3× (PD1), CD70 CAR+ T cells exhibited tumors regrowth post rechallenge,2 out of 5 mice treated with 4× (CD70,PD1), CD70 CAR+ T cells exhibitedtumors regrowth post rechallenge, while none of the mice treated with 3×(CD70), CD70 CAR+ T cells exhibited tumors regrowth post rechallenge.This trend continued, at day 70, 4 out of 5 mice treated with 3× (PD1),CD70 CAR⁺ T cells exhibited tumors regrowth post rechallenge, 4 out of 5mice treated with 4× (CD70, PD1), CD70 CAR⁺ T cells exhibited tumorsregrowth post rechallenge, while only one of the mice treated with 3×(CD70), CD70 CAR⁺ T cells exhibited a small tumor regrowth that began toappear at 34 days post rechallenge. Even out to day 91, only 1 of 5 micetreated with 3× (CD70), CD70 CAR⁺ T cells was starting to exhibit tumorregrowth, indicating that TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells retaina higher in vivo efficacy after re-exposure to tumor cells.

TABLE 16 Size of rechallenge tumors in untreated mice or mice treatedwith CD70 CAR T cells Tumor volume (mm³) at day post CAR T cell dosingDay Day CAR T treatment Subject Day 77 81 Day 84 88 Day 91 2KO, CD70 CART 1 1044 1265 1853 1927 2150 2 653 927 1040 1123 1256 3 899 1267 16031678 2167 4 701 1287 1672 1817 2490 5 689 1146 1423 1525 1901 3KO (PD1),CD70 1 385 692 983 1172 1369 CAR T 2 0 0 0 0 0 3 566 738 1030 1537 17404 712 1111 1337 1482 1832 5 632 778 1017 1129 1289 3KO (CD70), CD70 1 00 0 0 0 CAR T 2 34 56 75 104 135 3 0 0 0 0 0 4 0 0 0 0 0 5 0 0 0 0 04KO, CD70 CAR T 1 66 91 182 215 304 2 56 85 119 126 155 3 0 0 0 0 0 4 76175 218 256 316 5 35 30 51 58 63 No treatment 1 567 1263 1673 1751 20202 882 1214 1535 1609 2047 3 1158 1304 1676 1924 2389 4 295 391 667 7891078 5 707 1213 1676 1766 2056

Treatment in Large Tumor Model

The in vivo efficacy of anti-CD70 CAR T cells against larger renal cellcarcinoma tumors was investigated. As above, CRISPR/Cas9 and AAV6 wereused to create human T cells that lack expression of the TCR, β2M, CD70and/or PD1 with concomitant expression from the TRAC locus using a CARconstruct targeting CD70 (SEQ ID NO: 45). In this example activated Tcells were first electroporated with 2, 3 or 4 distinct Cas9:sgRNA RNPcomplexes containing sgRNAs targeting TRAC (SEQ ID NO: 40), β2M (SEQ IDNO: 41), PD1 (SEQ ID NO: 42), and CD70 (SEQ ID NO: 36 or 37). The DNAdouble stranded break at the TRAC locus was repaired by homologydirected repair with an AAV6-delivered DNA template (SEQ ID NO: 43)(encoding anti-CD70 CAR comprising the amino acid sequence of SEQ ID NO:5) containing right and left homology arms to the TRAC locus flanking achimeric antigen receptor cassette (−/+ regulatory elements for geneexpression).

The resulting modified T cells are 2× KO (TRAC−/β2M−), 3× KO(TRAC−/β2M−/PD1− or TRAC−/β2M−/CD70−) and 4× KO (TRAC−/β2M−/PD1−/CD70−)anti-CD70 CAR+ (with 41BB costimulatory domain) T cells. The ability ofthese anti-CD70 CAR+ T cells to ameliorate disease caused by aCD70+renal carcinoma cell line was evaluated in NOG mice using methodsemployed by Translational Drug Development, LLC (Scottsdale, Ariz.). Inbrief, 12, 5-8 week old female mice, CIEA NOG(NOD.Cg-Prkdc^(scid)I12rg^(tm1Sug)/JicTac) were individually housed inventilated microisolator cages, maintained under pathogen-freeconditions, 5-7 days prior to the start of the study. Mice received asubcutaneous inoculation of 5×10⁶ A498 renal carcinoma cells/mouse. Whenmean tumor size reached 125-175 mm³ (target of ˜150 mm³), the mice werefurther divided into 5 treatment groups as shown in Table 17. On day 1,treatment group 2 to 5 received a single 200 μl intravenous dose ofanti-CD70 CAR+ T cells according to Table 17.

TABLE 17 Treatment groups T cell treatment Group CAR-T A498 cells (i.v)N 1 None 5 × 10⁶ None 5 cells/mouse 2 2X KO, CD70 5 × 10⁶ 1 × 10⁷ 5 CAR+T cells cells/mouse cells/mouse 3 3X KO (PD1), 5 × 10⁶ 1 × 10⁷ 4 CD70CAR+ T cells cells/mouse cells/mouse 4 3X KO (CD70,) 5 × 10⁶ 1 × 10⁷ 5CD70 CAR+ T cells cells/mouse cells/mouse 5 4X KO (CD70, PD1), 5 × 10⁶ 1× 10⁷ 5 CD70 CAR+ T cells cells/mouse cells/mouse

Tumor volume was measured 2 times weekly from day of treatmentinitiation. By day 4 treatment only the 3× KO (TRAC−/β2M−/CD70−)anti-CD70 CAR+ T cells and 4× KO (TRAC−/β2M−/PD1−/CD70−) anti-CD70 CAR+T cells began to show a decrease in tumor volume (FIG. 27C). Incontrast, the tumor growth for animals treated with 2× KO (TRAC−/β2M−)anti-CD70 CAR+ T cells or 3× KO (TRAC−/β2M−/PD1−) anti-CD70 CAR+ T cellswas similar to the no treatment group. By day 23 treatment, the 3× KO(TRAC−/β2M−/CD70−) anti-CD70 CAR+ T cells completely eliminated CD70+kidney cancer tumors in vivo. By day 23 treatment, elimination of thetumors in response to the 4× KO (TRAC−/β2M−/PD1−/CD70−) anti-CD70 CAR+ Tcells was almost complete, with 4 of 5 mice exhibiting no detectablekidney cancer tumors in vivo.

These data show that 3× KO (TRAC−/β2M−/CD70−) anti-CD70 CAR+ T cells and4× KO (TRAC−/β2M−/PD1−/CD70−) anti-CD70 CAR+ T cells can significantlyregress large CD70+ kidney cancer tumors in vivo.

In Vivo Tumor Model for anti-BCMA CAR in context of PD1, CD70, and PD1with CD70 Knock Outs.

The efficacy of TRAC−/β2M−/anti-BCMA (4-1BB co-stim) CAR+ T cells,TRAC−/β2M−/PD−1−/anti-BCMA (4-1BB co-stim), TRAC−/β2M−/CD70−/anti-BCMA(4-1BB co-stim), and TRAC−/β2M−/PD−1−/CD70−/anti-BCMA (4-1BB co-stim)CAR+ T cells against the subcutaneous MM.1S tumor xenograft model in NOGmice was evaluated. In brief, 25, 5-8 week old female, CIEA NOG(NOD.Cg-Prkdc^(scid)I12rg^(tm1Sug)/JicTac) mice were individually housedin ventilated microisolator cages, maintained under pathogen-freeconditions, 5-7 days prior to the start of the study. On day 1, 25 micereceived a subcutaneous inoculation in the right flank of 5×10⁶ MM.1Scells in 50% Matrigel/mouse. When the mean tumor volume reached between75 and 125 mm³, the mice were divided into 5 treatment groups (N=5) anddosed with T cell populations comprising ˜50% anti-BCMA CAR⁺ T cells, asindicated in Table 18.

TABLE 18 Dosing # of T Cells Anti-BCMA Group CAR T Cell injected CAR+ Tcells N 1 N/A N/A N/A 5 2 TRAC-/β2M-/ 1 × 10⁷ 5 × 10⁶ 5 anti-BCMAcells/mouse (5 million) 3 TRAC-/β2M-/ 1 × 10⁷ 5 × 10⁶ 5 PD-1-/anti-BCMAcells/mouse (5 million) 4 TRAC-/β2M-/ 1 × 10⁷ 5 × 10⁶ 5 CD70-/anti-BCMAcells/mouse (5 million) 5 TRAC-/β2M-/ 1 × 10⁷ 5 × 10⁶ 5 PD-1-/CD70-/cells/mouse (5 million) anti-BCMA

Tumor volume and body weights were measured twice weekly and individualmice were euthanized when their tumor volume reached ≥2000 mm³.

By day 16, all treatment groups showed tumor regression from thestarting volumes while animals in the control group had tumors averaginggreater than 1500 mm³. By day 27, all animals in the control group hadreached the tumor volume endpoint of ≥2000 mm³ while all treatmentgroups had an average tumor volume less than 20 mm³ (FIG. 28A). On day45, all mice from each treatment group (Groups 2-5) were furthersubjected to a secondary tumor challenge (re-challenge). The micereceived a second subcutaneous inoculation in the left flank of 5×10⁶MM.1S cells in 50% Matrigel/mouse. A new group of control mice wereentered (N=5) and also received an inoculation of 5×10⁶ MM.1S cells in50% Matrigel/mouse in the left flank.

All mice were monitored for tumor growth in both the initial right flanktumor and the rechallenge tumor in the left flank. All treatment groupssuccessfully inhibited tumor growth in the initial right flank tumor inmost subjects (FIG. 28A; Table 19). Tumor growth was inhibited by alltreatments both before and after tumor re-challenge for the duration ofthe experiment to day 77. Only one subject treated withTRAC−/β2M−/CD70−/PD1−/anti-BCMA CAR⁺ T cells exhibited tumor growth fromthe initial cancer cell challenge (Table 19).

Surprisingly, tumor growth after re-challenge in the left flank was alsosignificantly inhibited by all treatment groups from the date ofre-challenge (day 45) to day 77 (FIG. 28B; Table 19). These datademonstrate that the CAR+ T cells persist in vivo to inhibit initialtumor growth, as well as inhibiting growth of new tumors following are-challenge with additional cancer cells even though no further CAR-Tcells were delivered to these mice. For example, three of the four miceinitially treated with populations of TRAC−/β2M−/anti-BCMA CAR⁺ T cells,three of the five mice initially treated with TRAC−/β2M−/CD70−/anti-BCMACAR⁺ T cells, and three of the five mice initially treated withTRAC−/β2M−/PD1−/anti-BCMA CAR⁺ T cells, exhibited no new tumor growthdespite a second challenge (re-challenge) to new cancer cells. Thesedata demonstrate that, unexpectedly, anti-BCMA CAR⁺ T cells are capableof persisting for long periods of time in vivo, e.g., up to at least 77days following injection, and retain their ability to inhibit tumor cellgrowth and reduce tumor volumes for long periods in vivo. Thesesurprising results indicate that use of such TRAC−/β2M−/anti-BCMA CAR⁺ Tcells would achieve superior long-term anti-cancer effect in vivo.

Human CD45+ cells were quantified from mouse blood using BD Trucounttubes following the manufacturers protocol and detected using BrilliantViolet 786 conjugated anti-human CD45 (Biolegend Cat #368528). Allgroups showed values of less than 100 huCD45+ cells/μl at 1 week. Twoweeks post dosing, the number of circulating CD45+ in all groups peakedbefore falling to pre-week 1 values by week 3 (FIG. 29). Uponre-challenge at Day 45, the anti-BCMA CAR+ T cell treated subjects wereable to eliminate or inhibit tumor growth without subsequent expansionof circulating CAR T cells following cancer rechallenge. These datafurther demonstrate that, unexpectedly, anti-BCMA CAR+ T cells arecapable of persisting for long periods of time in vivo, e.g., up to atleast 77 days following injection, and retain their ability to inhibittumor cell growth and reduce tumor volumes for long periods in vivo.These surprising results indicate that use of TRAC−/β2M−/anti-BCMA CAR+T cells, TRAC−/β2M−/CD70−/anti-BCMA CAR+ T cells andTRAC−/β2M−/PD1−/anti-BCMA CAR+ T cells would achieve superior long-termanti-cancer effect in vivo.

TABLE 19 Size of tumors in untreated mice or mice treated with anti-BCMACAR T cells Tumor volume Tumor volume at Day 45 (mm³) at Day 77 (mm³)Treatment Mouse Right Flank Right Flank Left Flank No Treatment 1 TS TSTS 2 TS TS TS 3 TS TS TS 4 TS TS TS 5 TS TS TS TRAC-/β2M-/ 1 0 0 (MS at0 (MS at anti-BCMA Day 59) Day 59:) 2 0 0 0 3 0 0 0 4 0 0 297 5 FD-T atFD-T at FD-T at day 16 day 16 day 16 TRAC-/ 1 0 0 1820 β2M-/PD1-/ 2 0 00 anti-BCMA 3 0 0 487 4 0 0 0 5 0 0 0 TRAC-/ 1 10  0 0 β2M-CD70-/ 2 0 00 anti-BCMA 3 0 0 (TS at 2349 (TS at day 77) day 77) 4 0 0 0 5 0 0 258TRAC-/β2M-/ 1 0 0 1157 PD1-/CD70-/ 2 0 0 1842 anti-BCMA 3 0 0 1664 4 0 00 5 89  1583 (TS at 1560 (TS at day 73) day 73) TS = sacrificed becauseof tumor volume; MS = Moribund sacrifice; FD-T = animal found dead.

In vivo Tumor Model for Anti-BCMA CAR in Context of CD70 Knockout:Effect of CD70 KO on Moderate CAR T Dosing

The efficacy of several anti-BCMA CAR⁺ T cell genotypes, both with andwithout CD70 knockouts, was evaluated against the subcutaneous RPMI-8226tumor xenograft model in NOG mice. In brief, eighty five (85), 5-8 weekold female, CIEA NOG (NOD.Cg-Prkdc^(scid)I12rg^(tm1Sug)/JicTac) micewere individually housed in ventilated microisolator cages, maintainedunder pathogen-free conditions, 5-7 days prior to the start of thestudy. On day 1 mice received a subcutaneous inoculation of 10×10⁶RPMI-8226 cells/mouse. Ten (10) days post inoculation with RPMI-8226cells, the mice were divided into 17 treatment groups (N=5) and dosedwith T cell populations comprising ˜80% anti-BCMA CAR⁺ T cells, asindicated in Table 20.

TABLE 20 Anti-BCMA # of T Cells Anti-BCMA Group CAR T Cell injected CAR+T cells N 1 N/A N/A N/A 5 2 TRAC-/β2M-/ 3 × 10⁶ 2.4 × 10⁶ 5 anti-BCMAcells/mouse (2.4 million) 3 TRAC-/β2M-/ 1 × 10⁶  8 × 10⁵ 5 anti-BCMAcells/mouse (0.8 million) 4 TRAC-/β2M-/ 3 × 10⁵ 2.4 × 10⁵ 5 anti-BCMAcells/mouse (0.24 million)  5 TRAC-/β2M-/ 1 × 10⁵  8 × 10⁴ 5 anti-BCMAcells/mouse (0.08 million)  6 TRAC-/β2M-/ 3 × 10⁶ 2.4 × 10⁶ 5PD1-/anti-BCMA cells/mouse (2.4 million) 7 TRAC-/β2M-/ 1 × 10⁶  8 × 10⁵5 PD1-/anti-BCMA cells/mouse (0.8 million) 8 TRAC-/β2M-/ 3 × 10⁵ 2.4 ×10⁵ 5 PD1-/anti-BCMA cells/mouse (0.24 million)  9 TRAC-/β2M-/ 1 × 10⁵ 8 × 10⁴ 5 PD1-/anti-BCMA cells/mouse (0.08 million)  10 TRAC-/β2M-/ 3 ×10⁶ 2.4 × 10⁶ 5 CD70-/anti-BCMA cells/mouse (2.4 million) 11 TRAC-/β2M-/1 × 10⁶  8 × 10⁵ 5 CD70-/anti-BCMA cells/mouse (0.8 million) 12TRAC-/β2M-/ 3 × 10⁵ 2.4 × 10⁵ 5 CD70-/anti-BCMA cells/mouse (0.24million)  13 TRAC-/β2M-/ 1 × 10⁵  8 × 10⁴ 5 CD70-/anti-BCMA cells/mouse(0.08 million)  14 TRAC-/β2M-/ 3 × 10⁶ 2.4 × 10⁶ 5 PD1-/CD70-/cells/mouse (2.4 million) anti-BCMA 15 TRAC-/β2M-/ 1 × 10⁶  8 × 10⁵ 5PD1-/CD70-/ cells/mouse (0.8 million) anti-BCMA 16 TRAC-/β2M-/ 3 × 10⁵2.4 × 10⁵ 5 PD1-/CD70-/ cells/mouse (0.24 million)  anti-BCMA 17TRAC-/β2M-/ 1 × 10⁵  8 × 10⁴ 5 PD1-/CD70-/ cells/mouse (0.08 million) anti-BCMA

Tumor volume and body weight was measured twice weekly, and individualmice were euthanized when tumor volume was ≥2000 mm³. By day 22, thedata show a statistically significant decrease in the tumor volume inresponse to higher doses of anti-BCMA CAR T cells (1×10⁵-3×10⁶ celldoses) compared to any anti-BCMA CART cell genotype dosed at 100,000cells (groups 5, 9, 13 and 17) (FIG. 30; Table 21).

At day 36, the TRAC−/β2M−/CD70−/anti-BCMA CAR+ T cells dosed at amoderate dose of 3×10⁵ cells exhibited a greater effect on decreasingtumor volume than the anti-BCMA CAR+ T cells without a CD70 KO (e.g.,TRAC−/β2M−/anti-BCMA CAR+ T cells, TRAC−/β2M−/PD1−/anti-BCMA CAR+ Tcells, or TRAC−/β2M−/PD1−/CD70−/anti-BCMA CAR+ T cells) (FIG. 30). Allof the higher doses of 1×10⁶ or greater all anti-BCMA CAR+ T cells (FIG.30; 1 Mil, 3 Mil), showed complete regression in tumor volume. Thistrend continued out to Day 57 of the study.

These results demonstrate that inhibiting the activity of CD70 (e.g., byknocking out CD70) increases the efficacy and potency of CAR⁺ T cells invivo. This effect is independent of the presence of an anti-CD70 CAR.

TABLE 21 Anti- BCMA CAR+ T cells/ Tumor Volume (mm³) Tumor Volume (mm³)Group Treatment dose at Day 36 at Day 57 1 No N/A 220 220 186 173 2781790 1794 938 2055 2029 Treatment 2 TRAC-/ 2.4 × 0 0 0 0 0 0 0 0 0 0β2M-/anti- 10⁶ BCMA (2.4 million) 3 8 × 0 0 0 0 0 0 0 0 0 0 10⁵ (0.8million) 4 2.4 × 65 65 77 56 0 516 441 257 97 337 10⁵ (0.24 million) 5 8× 264 264 386 276 185 2391 2283 2058 2147 2359 10⁴ (0.08 million) 6TRAC-/ 2.4 × 0 0 0 0 0 0 0 0 0 0 β2M-/ 10⁶ PD1-/anti- (2.4 BCMA million)7 8 × 0 0 0 0 0 0 0 0 0 0 10⁵ (0.8 million) 8 2.4 × 135 135 59 57 28 764518 280 181 79 10⁵ (0.24 million) 9 8 × 10⁴ 261 261 265 287 312 15321557 2218 2010 2098 (0.08 million) 10 TRAC-/ 2.4 × 0 0 0 0 0 0 0 0 0 0β2M-/ 10⁶ CD70-/ (2.4 anti- million) 11 BCMA 8 × 0 0 0 0 0 0 0 0 0 0 10⁵(0.8 million) 12 2.4 × 47 47 0 0 0 526 58 47 0 0 10⁵ (0.24 million) 13 8× 10⁴ 292 292 267 313 235 2075 2127 2096 1365 2354 (0.08 million) 14TRAC-/ 2.4 × 0 0 0 0 0 0 0 0 0 0 β2M-/ 10⁶ PD1-/ (2.4 CD70-/ million) 15anti- 8 × 0 0 0 0 0 0 0 0 0 0 BCMA 10⁵ (0.8 million) 16 2.4 × 100 100 9119 20 478 576 82 131 289 10⁵ (0.24 million) 17 8 × 310 310 319 345 4511528 2160 2843 2557 1499 10⁴ (0.08 million)

Example 9. Multi Knockout CAR T Cells Retain Cytokine Dependency

Cytokine Dependency. To determine whether gene editing resulted inunwanted off-target editing that could generate cells with adverseproperties, such as uncontrolled cell growth, the ability gene editedCAR T cells to grow in the absence of cytokines and/or serum wasassessed.

Anti-CD70 CAR T cells: The ability of TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺cells to grow in the absence of cytokines and/or serum was assessed.5×10⁶ TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ cells were plated ˜2 weeks postcell production (Day 0). The number of viable cells were enumerated 7and 14 days post plating in either full media, 5% human serum withoutcytokines (IL-2 and IL-7), or base media lacking serum and cytokines. Nocells were detected at 14 days plated in the cultures that lackedcytokines, suggesting that any potential off-target effects due togenome editing did not induce growth factor independentgrowth/proliferation to the cells (FIG. 31). The cells only proliferatedin the presence of cytokines (full media that contains cytokines) anddid not proliferate in the presence of serum alone. Thus, in vivo, thecells would likely not grow in the absence of cytokine, growth factor orantigen stimulation due to any off-target genome editing.

The ability of TRAC⁻/β2M⁻/CD70⁻/PD1⁻ anti-CD70 CAR⁺ cells to grow in theabsence of cytokines and/or serum was also assessed. 2×10⁶ cells wereplated ˜2 weeks post cell production (Day 0). The number of viable cellswere enumerated until 26 days post plating in either full media, 5%human serum without cytokines (IL-2 and IL-7), or base media lackingserum and cytokines. No cells were detected at 26 days plated in thecultures that lacked cytokines, suggesting that any potential off-targeteffects due to genome editing did not induce growth factor independentgrowth/proliferation to the cells (FIG. 32). The cells only proliferatedin the presence of cytokines (full media that contains cytokines) anddid not proliferate in the presence of serum alone. Thus, genome editingdid not induce any adverse events that allow the cells to grow in theabsence of cytokine, growth factor or antigen stimulation.

Anti-BCMA CAR+ T cells: The ability of TRAC⁻/β2M⁻/CD70⁻/PD-1⁻/anti-BCMACAR⁺ cells to grow in the absence of cytokines and/or serum wasassessed. TRAC⁻/β2M⁻/CD70⁻/PD-1⁻/anti-BCMA CAR⁺ cells are also referredto as 4× KO, BCMA CAR⁺ cells. 1×10⁶ 4× KO, BCMA CAR+ cells were platedfollowing the 10 rechallenges described in Example 6. The number ofviable cells were enumerated 7 and 14 days post plating in either fullmedia, 5% human serum without cytokines (IL-2 and IL-7), or base medialacking serum and cytokines. No cells were detected at 13 days plated inthe cultures that lacked cytokines, suggesting that any potentialoff-target effects due to genome editing did not induce growth factorindependent growth/proliferation to the cells (FIG. 33). The cells onlyproliferated in the presence of cytokines (full media that containscytokines) and did not proliferate in the presence of serum alone. Thus,in vivo, the cells would likely not grow in an uncontrolled way.

Other CAR T cells: It has previously been shown that the anti-CD33 CAR+T cells and anti-CD19 CAR+ T cells exemplified herein only proliferatedin the presence of cytokines and do not proliferate in the presence ofserum alone. Thus, in vivo, these cells would likely not grow in anuncontrolled way.

Cytokine Release Assay. To measure cytokine release, T cells and targetcells were co-incubated for 24 hours at the ratios indicated.Supernatant media was collected for use in IL-2 or IFNγ ELISAs (RDSystems) on a new plate following the manufacturer's instructions (RDSystems).

The ability of the TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ cells to produceinterleukin-2 (IL-2) when co-cultured in the presence of A498 cells wasanalyzed using the ELISA assay. Both the triple knockoutTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells and double knockoutTRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cells secreted high levels of IL-2.Strikingly, the TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ cells secreted higherlevels of IL-2 than the TRAC⁻/β2M⁻/anti-CD70 CAR⁺ cells when culturedwith A498 cells (FIG. 34). These results suggest that knocking-out theCD70 gene gives an advantage to anti-CD70 CAR+ T cells to secrete moreIL-2.

Example 10. Effect of Multiple Knockout on Anti-CD70 CAR+ T cells onA498 Renal Carcinoma Cells Effect of Multi Knock-Out on the Function ofAnti-CD70 CAR+ T Cells.

Cell Killing Assay. The ability of multi-gene editing to kill A498 renalcarcinoma cellswas determined using the cell kill assay described above.In brief, the TRAC⁻/β2M⁻/anti-CD70 CAR⁺ (2× KO, CD70 CAR⁺),TRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ (3× KO (PD-1), CD70 CAR⁺),TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ (3× KO (CD70), CD70 CAR⁺) andTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ (4× KO, CD70 CAR⁺) cells wereincubated with a CD70⁺ adherent RCC-derived cell line (A498 cells) atvarious CAR T cell:A498 target cells ratios.

The TRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ cells exhibited potent cellkilling of RCC-derived cells following 24-hour co-incubation (FIG. 35).The quadruple TRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T demonstratedhigher cell kill potency than triple knockout TRAC⁻/β2M⁻/PD-1⁻/anti-CD70CAR⁺ T cells that demonstrated higher potency than double knockoutTRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cells (visible at low T-cell: A498 Ratio of0.5:1 and 0.25:1). The results demonstrate knocking out both the CD70and PD-1 genes gave the anti-CD70 CAR+ cells higher cell kill potency.

The gene edited cells also exhibited potent cell killing of RCC-derivedcells following 24-hour co-incubation at a CAR T cell:A948 target cellratio of 0.24:1. (FIG. 36). Specifically, the triple knockoutTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells and the quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells demonstrated higherpotency than the double knockout TRAC⁻/β2M⁻/anti-CD70 CAR⁺ T cells orthe triple knockout TRAC⁻/β2M⁻/PD-1⁻/anti-CD70 CAR⁺ T cells. These dataindicate that knockout of CD70 in the context of an anti-CD70 CARimproved the cell killing ability of the anti-CD70 CAR⁺ T cells.

Cytokine Release Assay. A cytokine release assay was performed asdescribed above. The ability of the double knockout, triple knockout,and quadruple knockout anti-CD70 CAR⁺ T cells to produce IL-2 andinterferon gamma (IFN-gamma (IFN-g)) when co-cultured in the presence ofA498 cells following 24-hour co-incubation at a ratio (CAR T cell:A498target cell) of 0.25:1 was assessed using an ELISA assay. IL-2 and IFN-gfrom supernatants of cell co-cultures were measured. The triple knockoutTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells and quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells secreted the highestlevels of IFN-g (FIG. 37A) and IL-2 (FIG. 37B) when cultured with A498cells.

Effect of CD70 Knockout on Exhaustion Marker Expression

The levels of the exhaustion markers PD-1 and LAG3 were assessed on theTRAC⁻/β2M⁻/anti-CD70 CAR⁺ (2× KO, CD70 CAR⁺), TRAC⁻/β2M⁻/PD-1⁻/anti-CD70CAR⁺ (3× KO (PD-1), CD70 CAR⁺), TRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ (3× KO(CD70), CD70 CAR⁺) and TRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ (4× KO,CD70 CAR⁺) T cells used in the Examples above. CD4⁺ T cells wereassessed for PD-1 expression (FIG. 38) and both CD8⁺ T cells and CD4⁺ Tcells were assessed for LAG3 expression (FIG. 39A and FIG. 39B,respectively) by flow cytometry.

The data demonstrate that CD70 KO reduces exhaustion marker expressionin CAR T cells. The data in FIG. 38 shows that PD-1 expression isdecreased, as expected, when PD-1 is knocked out, and it is alsodecreased when CD70 is knocked out.

The data in FIGS. 39A and 39B show that knocking out CD70, reduces theLAG3 expression marker in CD4 and CD8 cells.

The data demonstrate that knocking out CD70, specifically, could reducethe potential exhaustion of the CD8⁺ and CD4⁺ gene edited populations ofCAR+ T cells leading to better therapeutics.

Example 11. CD70 KO Improves Cell Kill in Multiple Cell Types

CD70 Expression in Various Cancer Cell Lines. Relative CD70 expressionwas measured in various cancer cell lines to further evaluate theability of anti-CD70 CAR⁺ T cells to kill various cancer types. CD70expression was measured by FACS analysis using Alexa Fluor 647anti-human CD70 antibody (BioLegend Cat. No. 355115). FIG. 40A (leftgraph) shows the relative expression of CD70 in ACHN cells, as measuredby FACS, compared to other kidney cancer cell lines A498, 786-O, cacki-1and Caki-2. Additionally, non-kidney cancer cell lines were evaluatedfor CD70 expression by FACS analysis (Table 22, FIG. 40A and FIG. 40B)using either an Alexa Fluor 647 anti-human CD70 antibody (BioLegend Cat.No. 355115; FIG. 40A, right panel) or a FITC anti-human CD70 antibody(BioLegend Cat. No. 355105) in FIG. 40B. SNU-1 (intestinal cancer cells)exhibited high levels of CD70 expression that were similar to A498 (FIG.40A, right panel). SKOV-3 (ovarian), HuT78 (lymphoma), NCI-H1975 (lung)and Hs-766T (pancreatic) cell lines exhibited levels of CD70 expressionthat were similar or higher than ACHN but lower than A498 (Table 22,FIG. 40B).

TABLE 22 Relative CD70 Cell Line Cancer type expression A498 KidneyCarcinoma High ACHN Kidney (derived from Medium-Low metastasis) SK-OV-3Ovarian Adenocarcinoma Medium NCI-H1975 Lung Adenocarcinoma (NSCLC)Medium Calu-1 Lung Carcinoma Low DU 145 Prostate Carcinoma Low SNU-1Gastric Carcinoma High Hs 766T Pancreatic Carcinoma Medium MJ T cellLymphoma High HuT78 T cell Lymphoma Medium HuT102 T cell Lymphoma MediumPANC-1 Pancreatic Carcinoma Low U937 AML No expression K562 chronicmyelogenous leukemia No expression (Negative Control)

Cell Kill Assay. The ability of multi-gene edited anti-CD70 CAR+ cellsto kill ACHN renal carcinoma cells was determined using the cell killassay described above. The TRAC⁻/β2M⁻/anti-CD70 CAR⁺ (2× KO, CD70 CAR⁺),TRAC⁻/β2M⁻/anti-CD70 CAR⁺ (3× KO (PD-1), CD70 CAR⁺),TRAC⁻/β2M⁻/anti-CD70⁻/anti-CD70 CAR⁺ (3× KO (CD70), CD70 CAR⁺) andTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ (4× KO, CD70 CAR⁺) cells were incubatedwith an adherent RCC-derived cell line expressing low levels of CD70antigen (ACHN cells) (FIG. 40A shows the relative expression of CD70 inACHN cells, as measured by FACS, compared to other kidney cancer celllines A498, 786-O,l cacki-1 and Caki-2) at a CAR T cell:ACHN targetcells ratio of 0.5:1 (FIG. 40C) and 0.25:1 (FIG. 40D). The gene editedcells exhibited potent cell killing of RCC-derived cells following24-hour co-incubation (FIGS. 40C and 40D). The cells demonstrated higherpotency when PD-1 was knocked out, when CD70 was knocked out, and evenslightly higher potency when both PD-1 and CD70 were knocked out. Inconclusion, knockout of PD-1 or CD70 or of both PD-1 and CD70 togetherimproves the cell killing ability of the anti-CD70 CAR+ cells in ACHNcells.

Although ACHN cells were found to express moderate to low levels ofCD70, they were surprisingly susceptible to killing by 3× KO (PD-1),CD70 CAR+ T cells, 3× KO (CD70), CD70 CAR+ T cells, and 4× KO CD70 CAR+T cells (FIG. 40C and 40D). This indicates that high CD70 expression isnot a requirement for effective killing of a target cell by gene-editedT cells that express an anti-CD70 CAR. Additionally, given that thelevels of CD70 expression on SNU-1, SK-OV-3, NCI-H1975 and HS-766T celllines were found to be similar or higher than ACHN, it was expected thatanti-CD70 CAR⁺ T cells would be especially efficient at killing thesecancer cell types as well. Indeed, it was found thatTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ (4× KO, CD70 CAR⁺) andTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ (3× KO (CD70), CD70 CAR⁺) exhibitedsurprisingly potent cell killing of numerous solid tumor cell linesafter only 24 hours of co-culture (FIG. 40E shows killing by 4× KO CAR+T cells and FIG. 40F shows killing by 3× KO CAR+ T cells). Both 3× KO,CD70 CAR+ and 4× KO, CD70 CAR+ T cells killed >60% of kidney,pancreatic, and ovarian tumor cells (A498, ACHN, SK-OV-3, and Hs-766T)at a 4:1 effector:target cell ratio and >50% at a 1:1 effector:targetcell ratio. Cell killing of cancer cell lines that had medium to lowCD70 expression (NCI-H1975, Calu-1 and DU 145) was still effectivewith >30% killing at an effector:target cell ratio of 4:1 within 24hours of co-culture (FIGS. 40E and 40F). Longer exposure (i.e., 96hours) to either 3× KO or 4× KO, CD70 CAR+ T cells resulted in anincrease in cancer cell killing across all cell types, particularly forSKOV-3, Hs-766T, and NIC-H1975 cells wherein killing was >80% at aneffector:target cell ratio of 1:1 (FIG. 40G).

SNU-1 Cell Kill by was Assessed by Visual Assessment.

Target cell killing following long exposure to CAR+ T cells was alsoassessed by microscopy for SNU-1 cancer cells. SNU-1 cells were platedat a density of 1 million cells per well in a 6 well plate and mixed atan effector:target ratio of 4:1 with 3× KO (CD70), anti-CD70 CAR⁺ Tcells. The co-culture was incubated for six (6) days and the presence ofviable cancer cells was assessed by microscope. All gastric carcinomatarget cells (SNU-1) were eliminated in wells containingTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells, as compared to control wells,indicating cancer cells were completely eliminated by anti-CD70 CAR⁺ Tcells with an extended co-culture.

The ability of anti-CD70 CAR+ T cells to selectively killCD70-expressing cells was determined. A flow cytometry assay wasdesigned to test killing of cancer cell suspension lines (e.g., K562,MM.1S and HuT78 cancer cells that are referred to as “target cells”) by3× KO (CD70) (TRAC⁻/β2M⁻/CD70⁻) anti-CD70 CAR+ T cells. Two of thetarget cell lines that were used were CD70-expressing cancer cells(e.g., MM.1S and HuT78), while a third that was used as negative controlcancer cells lack CD70 expression (e.g., K562). TheTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR+ T cells were co-cultured with either theCD70-expressing MM.1S or HuT78 cell lines or the CD70-negative K562 cellline. The target cells were labeled with 5 μM efluor670 (eBiosciences),washed and seeded at a density of 50,000 target cells per well in a96-well U-bottom plate. The target cells were co-cultured withTRAC⁻/β2M⁻/CD70⁻ anti-CD70 CAR+ T cells at varying ratios (0.5:1, 1:1,2:1 and 4:1 CAR+ T cells to target cells) and incubated overnight.Target cell killing was determined following a 24 hour co-culture. Thecells were washed and 200 μL of media containing a 1:500 dilution of 5mg/mL DAPI (Molecular Probes) (to enumerate dead/dying cells) was addedto each well. Cells were then analyzed by flow cytometry and the amountof remaining live target cells was quantified.

FIG. 40H, FIG. 40I, and FIG. 40J demonstrate selective target cellkilling by TRAC−/B2M−/CD70− anti-CD70 CAR+ T cells. A 24 hour co-culturewith 3× KO (CD70) CAR+ T cells resulted in nearly complete killing of Tcell lymphoma cells (HuT78), even at a low CAR+ T cell toCD70-expressing target cell ratio of 0.5:1 (FIG. 40J) Likewise, a 24hour co-culture resulted in nearly complete killing of multiple myelomacells (MM.1S) at all CAR+ T cell to target cell ratios tested (FIG.40I). Killing of target cells was found to be selective in thatTRAC−/B2M−/anti-CD70 CAR+ T cells induced no killing of CD70-deficientK562 cells that was above the level of control samples (e.g., eithercancer cells alone or co-culture with no RNP T cells) at anyeffector:target cell ratio tested (FIG. 40H).

Cytokine Release Assay. A cytokine release assay was performed asdescribed above. The ability of the double knockout, triple knockout,and quadruple knockout anti-CD70 CAR⁺ T cells to produce IL-2 and IFN-gwhen co-cultured in the presence of ACHN cells following 24-hourco-incubation at a ratio (CAR T cell:ACHN target cell) of 0.25:1 wasassessed using an ELISA assay. IL-2 and IFN-g from supernatants of cellco-cultures were measured. The triple knockoutTRAC⁻/β2M⁻/CD70⁻/anti-CD70 CAR⁺ T cells and quadruple knockoutTRAC⁻/β2M⁻/PD-1⁻/CD70⁻/anti-CD70 CAR⁺ T cells secreted the highestlevels of IFN-g (FIG. 41) and IL-2 (FIG. 41B) when cultured with ACHNcells. In conclusion, knockout of CD70 or of both PD-1 and CD70 togetherimproves the cell killing ability of the anti-CD70 CAR+ cells in ACHNcells.

Example 12. Efficacy of CD70 KO in Anti-CD70 CAR+ T Cells: The TumorXenograft Model in NOG Mice Treatment in the Ovarian Tumor Model

The ability of T cells expressing an anti-CD70 CAR to eliminate ovarianadenocarcinoma cells that express moderate levels of CD70 was evaluatedin vivo using a subcutaneous ovarian carcinoma (SKOV-3) tumor xenograftmodel in mice.

CRISPR/Cas9 and AAV6 were used as above (see for example, Example 3) togenerate human T cells that lack expression of the TCR, β2M, CD70 withconcomitant expression from the TRAC locus using a CAR constructtargeting CD70 (SEQ ID NO: 45; SEQ ID NO: 46. In this example activatedT cells were first electroporated with 3 distinct Cas9:sgRNA RNPcomplexes containing sgRNAs targeting TRAC (SEQ ID NO: 40), β2M (SEQ IDNO: 41), and CD70 (SEQ ID NO: 36 or 37). The DNA double stranded breakat the TRAC locus was repaired by homology directed repair with anAAV6-delivered DNA template comprising a donor template (SEQ ID NO: 44;SEQ ID NO: 45) (encoding anti-CD70 CAR comprising the amino acidsequence of SEQ ID NO: 45) containing right and left homology arms tothe TRAC locus flanking a chimeric antigen receptor cassette (−/+regulatory elements for gene expression).

The resulting modified T cells are 3× KO (TRAC−/β2M−/CD70−) anti-CD70CAR+ T cells. The ability of these anti-CD70 CAR+ T cells to amelioratedisease caused by a CD70+ ovarian carcinoma cell line was evaluated inNOG mice using methods employed by Translational Drug Development, LLC(Scottsdale, Ariz.). In brief, 12 5-8 week old female, CIEA NOG(NOD.Cg-Prkd^(scid)I12rg^(tm1Sug)/JicTac) mice were individually housedin ventilated microisolator cages, maintained under pathogen-freeconditions, 5-7 days prior to the start of the study. Mice received asubcutaneous inoculation of 5×10⁶ SKOV-3 ovarian carcinoma cells/mousein the right hind flank. When mean tumor size reached 25-75 mm³ (targetof ˜50 mm³), the mice were further divided into two treatment groups asshown in Table 23. On Day 1, treatment group 2 received a single 200 μlintravenous dose of anti-CD7OCAR+ T cells according to Table 23.

TABLE 23 Treatment groups T cell SKOV-3 treatment Group CAR-T cells(i.v.) N 1 None 5 × 10⁶ None 5 cells/mouse 2 3X KO (CD70,) 5 × 10⁶ 1 ×10⁷ 5 anti-CD70 CAR+ T cells cells/mouse cells/mouse

Tumor volume was measured 2 times weekly from day of treatmentinitiation. By day 9 post-injection, tumors treated with anti-CD70 CARTcells began to show a decrease in tumor volume relative to tumors inuntreated animals. By day 17 post-injection, CD70+ ovarian cancer tumorsin mice treated with anti-CD70 CAR T cells were completely eliminated.This complete regression of tumor growth was sustained in treatedanimals through day 44 post-injection, whereupon 4 out of 5 mice treatedwith anti-CD70 CART cells remained tumor-free until theend-of-observation (day 69) (FIG. 42A). These data demonstrate that 3×KO (TRAC−/β2M−/CD70−) anti-CD70 CAR+ cells are highly potent in vivo fortreating human ovarian tumors.

Treatment in the Non-Small Cell Lung Carcinoma (NSCLC) Tumor Model

The ability of T cells expressing a CD70 CAR to eliminate lungadenocarcionma cells that express moderate levels of CD70 was evaluatedin in vivo using a subcutaneous lung carcinoma (NCI-H1975) tumorxenograft model in mice.

CRISPR/Cas9 and AAV6 were used as above (see for example, Example 3) tocreate human T cells that lack expression of the TCR, β2M, CD70 withconcomitant expression from the TRAC locus using a CAR constructtargeting CD70 (SEQ ID NO: 43; SEQ ID NO: 44). In this example activatedT cells were first electroporated with 3 distinct Cas9:sgRNA RNPcomplexes containing sgRNAs targeting TRAC (SEQ ID NO: 40), β 2M (SEQ IDNO: 41), and CD70 (SEQ ID NO: 36 or 37). The DNA double stranded breakat the TRAC locus was repaired by homology directed repair with anAAV6-delivered DNA template (SEQ ID NO: 43; SEQ ID NO: 44) (encodinganti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 45)containing right and left homology arms to the TRAC locus flanking achimeric antigen receptor cassette (−/+ regulatory elements for geneexpression).

The resulting modified T cells are 3× KO (TRAC−/(β2M−/CD70−) anti-CD70CAR+ (with 41BB costimulatory domain) T cells. The ability of theseanti-CD70 CAR+ T cells to ameliorate disease caused by a CD70+ lungcarcinoma cell line was evaluated in NOG mice using methods employed byTranslational Drug Development, LLC (Scottsdale, Ariz.). In brief, 12,5-8 week old female, CIEA NOG (NOD.Cg-Prkdc^(scid)I12rg^(tm1Sug)/JicTac)mice were individually housed in ventilated microisolator cages,maintained under pathogen-free conditions, 5-7 days prior to the startof the study. Mice received a subcutaneous inoculation of 5×10⁶NCI-H1975 lung carcinoma cells/mouse in the right hind flank. When meantumor size reached 25-75 mm³ (target of ˜50 mm³), the mice were furtherdivided into 2 treatment groups as shown in Table 24. On Day 1,treatment group 2 received a single 200 μl intravenous dose ofanti-CD7OCAR+ T cells according to Table 24.

TABLE 24 Treatment groups T cell NCI-H1975 treatment Group CAR-T cells(i.v.) N 1 None 5 × 10⁶ None 5 cells/mouse 2 3X KO (CD70,) 5 × 10⁶ 1 ×10⁷ 5 anti-CD70 CAR+ T cells cells/mouse cells/mouse

Tumor volume was measured 2 times weekly from day of treatmentinitiation. By day 12 post-injection, tumors treated with anti-CD70 CART cells began to show a decrease in tumor volume relative to tumors inuntreated animals. This complete regression of tumors in treated animalscontinue through day 33 post injection. Treatment with anti-CD70 CAR Tcells resulted in potent activity against established H1975 lung cancerxenografts through 40 days post injection (tumor regrowth was suppressedin all mice up to day 40 with tumor size <100 mm³), whereupon tumorsbegan to grow. (FIG. 42B). These data demonstrate that 3× KO(TRAC−/β2M−/CD70−) anti-CD70 CAR+ cells have potent activity againsthuman CD70+lung cancer tumors in vivo.

Treatment in the Pancreatic Tumor Model

The ability of T cells expressing a CD70 CAR to eliminate pancreaticcarcinoma cells that express moderate levels of CD70 was evaluated in invivo using a subcutaneous pancreatic (Hs 766T) tumor xenograft model inmice.

CRISPR/Cas9 and AAV6 were used as above (see for example, Example 3) tocreate human T cells that lack expression of the TCR, β2M, CD70 withconcomitant expression from the TRAC locus using a CAR constructtargeting CD70 (SEQ ID NO: 43; SEQ ID NO: 44). In this example activatedT cells were first electroporated with 3 distinct Cas9:sgRNA RNPcomplexes containing sgRNAs targeting TRAC (SEQ ID NO: 40), β2M (SEQ IDNO: 41), and CD70 (SEQ ID NO: 36 or 37). The DNA double stranded breakat the TRAC locus was repaired by homology directed repair with anAAV6-delivered DNA template (SEQ ID NO: 43; SEQ ID NO: 44) (encodinganti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 45)containing right and left homology arms to the TRAC locus flanking achimeric antigen receptor cassette (−/+ regulatory elements for geneexpression).

The resulting modified T cells are 3× KO (TRAC−/β2M−/CD70−) anti-CD70CAR+ T cells. The ability of these anti-CD70 CAR+ T cells to amelioratedisease caused by a CD70+ pancreatic carcinoma cell line was evaluatedin NOG mice using methods employed by Translational Drug Development,LLC (Scottsdale, Ariz.). In brief, 12, 5-8 week old female, CIEA NOG(NOD.Cg-Prkdc^(scid)I12rg^(tm1Sug)/JicTac) mice were individually housedin ventilated microisolator cages, maintained under pathogen-freeconditions, 5-7 days prior to the start of the study. Mice received asubcutaneous inoculation of 5×10⁶ Hs766T pancreatic carcinoma cells inthe right hind flank. When mean tumor size reached 25-75 mm³ (target of˜50 mm³), the mice were further divided into 2 treatment groups as shownin Table 25. On Day 1, treatment group 2 received a single 200 μlintravenous dose of anti-CD70 CAR+ T cells according to Table 25.

TABLE 25 Treatment groups T cell Hs766T treatment Group CAR-T cells(i.v.) N 1 None 5 × 10⁶ None 5 cells/mouse 2 3X KO (CD70,) 5 × 10⁶ 1 ×10⁷ 5 anti-CD70 CAR+ T cells cells/mouse cells/mouse

Tumor volume was measured 2 times weekly from day of treatmentinitiation. By Day 15 post-injection, tumors treated with anti-CD70 CART cells began to show a decrease in tumor volume in all treated mice.Treatment with anti-CD70 CAR+ T cells effectively reduced the size ofthe CD70+ pancreatic cancer tumors, in all mice tested (<37 mm³) with noevidence of further growth for the duration of the study (through Day67) (FIG. 42C). These data demonstrate that 3× KO (TRAC−/β2M−/CD70−)anti-CD70 CAR+ cells induce regression of human CD70+ pancreatic cancertumors in vivo, with potent activity against established Hs766Tpancreatic cancer xenografts and durable responses beyond 60 daysfollowing treatment initiation.

Treatment in the cutaneous T-cell Lymphoma Tumor Xenograft Model

The ability of T cells expressing an anti-CD70 CAR to eliminate T celllymphoma was evaluated in in vivo using a subcutaneous T-cell lymphoma(Hu T78) tumor xenograft model in mice.

CRISPR/Cas9 and AAV6 were used as above (see for example, Example 3) tocreate human T cells that lack expression of the TCR, β2M, CD70 withconcomitant expression from the TRAC locus using a CAR constructtargeting CD70 (SEQ ID NO: 43; SEQ ID NO: 44). In this example activatedT cells were first electroporated with 3 distinct Cas9:sgRNA RNPcomplexes containing sgRNAs targeting TRAC (SEQ ID NO: 40), β2M (SEQ IDNO: 41), and CD70 (SEQ ID NO: 36 or 37). The DNA double stranded breakat the TRAC locus was repaired by homology directed repair with anAAV6-delivered DNA template (SEQ ID NO: 43; SEQ ID NO: 44) (encodinganti-CD70 CAR comprising the amino acid sequence of SEQ ID NO: 45)containing right and left homology arms to the TRAC locus flanking achimeric antigen receptor cassette (−/+ regulatory elements for geneexpression).

The resulting modified T cells are 3× KO (TRAC−/β2M−/CD70−) anti-CD70CAR+ T cells. The ability of these anti-CD70 CAR+ T cells to amelioratedisease caused by a CD70+ T-cell lymphoma cell line was evaluated in NOGmice using methods employed by Translational Drug Development, LLC(Scottsdale, Ariz.). In brief, 12, 5-8 week old female, CIEA NOG(NOD.Cg-Prkdc^(scid)I12rg^(tm1Sig)/JicTac) mice were individually housedin ventilated microisolator cages, maintained under pathogen-freeconditions, 5-7 days prior to the start of the study. Mice received asubcutaneous inoculation of 3×10⁶ HuT78 T-cell lymphoma cells in theright hind flank. When mean tumor size reached 25-75 mm³ (target of ˜50mm³), the mice were further divided into 2 treatment groups as shown inTable 26. On Day 1, treatment group 2 received a single 200 μlintravenous dose of anti-CD70 CAR+ T cells according to Table 26.

TABLE 26 Treatment groups T cell HuT78 treatment Group CAR-T cells(i.v.) N 1 None 3 × 10⁶ None 5 cells/mouse 2 3X KO (CD70,) 3 × 10⁶ 1 ×10⁷ 4 anti-CD70 CAR+ T cells cells/mouse cells/mouse

Tumor volume was measured 2 times weekly from day of treatmentinitiation. By Day 12 post-injection, tumors treated with anti-CD70 CART cells began to show a decrease in tumor volume in all treated mice.Treatment with anti-CD70 CAR+ T cells effectively reduced the size ofthe CD70+ T-cell lymphoma tumors, in all mice tested at Day 15 (FIG.42C). These data demonstrate that 3× KO (TRAC−/β2M−/CD70−) anti-CD70CAR+ cells induce regression of human CD70+T-cell lymphoma tumors invivo, with potent activity against established HuT78 T-cell lymphomaxenografts.

Summary of Sequences SEQ ID NO Description Sequence 1 TRAC IndelAAGAGCAACAAATCTGACT 2 TRAC IndelAAGAGCAACAGTGCTGTGCCTGGAGCAACAAATCTGACTAAGAGCA ACAAATCTGACT 3 TRAC IndelAAGAGCAACAGTGCTGGAGCAACAAATCTGACTAAGAGCAACAAAT CTGACT 4 TRAC IndelAAGAGCAACAGTGCCTGGAGCAACAAATCTGACTAAGAGCAACAAA TCTGACT 5 TRAC IndelAAGAGCAACAGTGCTGACTAAGAGCAACAAATCTGACT 6 TRAC IndelAAGAGCAACAGTGCTGTGGGCCTGGAGCAACAAATCTGACTAAGAG CAACAAATCTGACT 7TRAC Indel AAGAGCAACAGTGCTGGCCTGGAGCAACAAATCTGACTAAGAGCAA CAAATCTGACT 8TRAC Indel AAGAGCAACAGTGCTGTGTGCCTGGAGCAACAAATCTGACTAAGAG CAACAAATCTGACT9 B2M Indel CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 10 B2M IndelCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 11 B2M IndelCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 12 B2M IndelCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGATAGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 13 B2M IndelCGTGGCCTTAGCTGTGCTCGCGCTATCCAGCGTGAGTCTCTCCTACC CTCCCGCT 14 B2M IndelCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGTGGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 15 sgRNAnnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu 16 sgRNAnnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc 17 sgRNAn₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcu₍₁₋₈₎ 18 4-1BBAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTA nucleotideTGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCG sequenceATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG 19 4-1BBKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL amino acid sequence 20CD28 amino SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS acid sequence 21CD3-z CGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAG nucleotideGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGA sequenceGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAG GCCCTGCCTCCCAGA 22 CD3-zRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG amino acidKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS sequenceTATKDTYDALHMQALPPR 23 CD70UCACCAAGCCCGCGACCAAUguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU (E1_T1) 24 CD70AUCACCAAGCCCGCGACCAAguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU (E1_T3) 25 CD70CGGUGCGGCGCAGGCCCUAUguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU (E1_T4) 26 CD70GCUUUGGUCCCAUUGGUCGCguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU (E1_T7) 27 CD70GCCCGCAGGACGCACCCAUAguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU (E1_T8) 28 CD70GUGCAUCCAGCGCUUCGCACguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU (E1_T10) 29 CD70CAGCUACGUAUCCAUCGUGAguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU (E3_T1) 30 TRACAGAGCAACAGUGCUGUGGCCguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU 31 β2MGCUACUCUCUCUUUCUGGCCguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU 32 PD-1CUGCAGCUUCUCCAACACAUguuuuagagcuagaaauagcaaguuaaaauaaggcuag sgRNAuccguuaucaacuugaaaaaguggcaccgagucggugcUUUU 33 CD70U*C*A*CCAAGCCCGCGACCAAUguuuuagagcuagaaauagcaaguuaaaauaaggc sgRNAuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U (E1_T1) 34 CD70A*U*C*ACCAAGCCCGCGACCAAguuuuagagcuagaaauagcaaguuaaaauaaggc sgRNAuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U (E1_T3) 35 CD70C*G*G*UGCGGCGCAGGCCCUAUguuuuagagcuagaaauagcaaguuaaaauaaggc sgRNAuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U (E1_T4) 36 CD70G*C*U*UUGGUCCCAUUGGUCGCguuuuagagcuagaaauagcaaguuaaaauaaggc sgRNAuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U (E1_T7) 37 CD70G*C*C*CGCAGGACGCACCCAUAguuuuagagcuagaaauagcaaguuaaaauaaggc sgRNAuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U (E1_T8) 38 CD70G*U*G*CAUCCAGCGCUUCGCACguuuuagagcuagaaauagcaaguuaaaauaaggc sgRNAuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U (E1_T10) 39 CD70C*A*G*CUACGUAUCCAUCGUGAguuuuagagcuagaaauagcaaguuaaaauaaggc sgRNAuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U (E3_T1) 40 TRACA*G*A*GCAACAGUGCUGUGGCCguuuuagagcuagaaauagcaaguuaaaauaagg sgRNAcuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U 41 β2MG*C*U*ACUCUCUCUUUCUGGCCguuuuagagcuagaaauagcaaguuaaaauaaggc sgRNAuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U 42 PD-1C*U*G*CAGCUUCUCCAACACAUguuuuagagcuagaaauagcaaguuaaaauaaggc sgRNAuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U*U*U 43 CD70CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGC rAAVGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCG (CD70BCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGC scFV withACGCGTGAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATAT 41BB)CGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGCAGGTCCAGTTGGTGCAAAGCGGGGCGGAGGTGAAAAAACCCGGCGCTTCCGTGAAGGTGTCCTGTAAGGCGTCCGGTTATACGTTCACGAACTACGGGATGAATTGGGTTCGCCAAGCGCCGGGGCAGGGACTGAAATGGATGGGGTGGATAAATACCTACACCGGCGAACCTACATACGCCGACGCTTTTAAAGGGCGAGTCACTATGACGCGCGATACCAGCATATCCACCGCATACATGGAGCTGTCCCGACTCCGGTCAGACGACACGGCTGTCTACTATTGTGCTCGGGACTATGGCGATTATGGCATGGACTACTGGGGTCAGGGTACGACTGTAACAGTTAGTAGTGGTGGAGGCGGCAGTGGCGGGGGGGGAAGCGGAGGAGGGGGTTCTGGTGACATAGTTATGACCCAATCCCCAGATAGTTTGGCGGTTTCTCTGGGCGAGAGGGCAACGATTAATTGTCGCGCATCAAAGAGCGTTTCAACGAGCGGATATTCTTTTATGCATTGGTACCAGCAAAAACCCGGACAACCGCCGAAGCTGCTGATCTACTTGGCTTCAAATCTTGAGTCTGGGGTGCCGGACCGATTTTCTGGTAGTGGAAGCGGAACTGACTTTACGCTCACGATCAGTTCACTGCAGGCTGAGGATGTAGCGGTCTATTATTGCCAGCACAGTAGAGAAGTCCCCTGGACCTTCGGTCAAGGCACGAAAGTAGAAATTAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGGTAACCACGTGCGGACCGAGGCTGCAGCGTCGTCCTCCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 44 CD70GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTA LHA toAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCA RHAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTT (CD70BCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCC scFV withCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTG 41BB)CTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGCAGGTCCAGTTGGTGCAAAGCGGGGCGGAGGTGAAAAAACCCGGCGCTTCCGTGAAGGTGTCCTGTAAGGCGTCCGGTTATACGTTCACGAACTACGGGATGAATTGGGTTCGCCAAGCGCCGGGGCAGGGACTGAAATGGATGGGGTGGATAAATACCTACACCGGCGAACCTACATACGCCGACGCTTTTAAAGGGCGAGTCACTATGACGCGCGATACCAGCATATCCACCGCATACATGGAGCTGTCCCGACTCCGGTCAGACGACACGGCTGTCTACTATTGTGCTCGGGACTATGGCGATTATGGCATGGACTACTGGGGTCAGGGTACGACTGTAACAGTTAGTAGTGGTGGAGGCGGCAGTGGCGGGGGGGGAAGCGGAGGAGGGGGTTCTGGTGACATAGTTATGACCCAATCCCCAGATAGTTTGGCGGTTTCTCTGGGCGAGAGGGCAACGATTAATTGTCGCGCATCAAAGAGCGTTTCAACGAGCGGATATTCTTTTATGCATTGGTACCAGCAAAAACCCGGACAACCGCCGAAGCTGCTGATCTACTTGGCTTCAAATCTTGAGTCTGGGGTGCCGGACCGATTTTCTGGTAGTGGAAGCGGAACTGACTTTACGCTCACGATCAGTTCACTGCAGGCTGAGGATGTAGCGGTCTATTATTGCCAGCACAGTAGAGAAGTCCCCTGGACCTTCGGTCAAGGCACGAAAGTAGAAATTAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG 45 CD70 CARATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCT nucleotideCCACGCAGCAAGGCCGCAGGTCCAGTTGGTGCAAAGCGGGGCGGAG sequenceGTGAAAAAACCCGGCGCTTCCGTGAAGGTGTCCTGTAAGGCGTCCG (CD70BGTTATACGTTCACGAACTACGGGATGAATTGGGTTCGCCAAGCGCCG scFV withGGGCAGGGACTGAAATGGATGGGGTGGATAAATACCTACACCGGCG 41BB)AACCTACATACGCCGACGCTTTTAAAGGGCGAGTCACTATGACGCGCGATACCAGCATATCCACCGCATACATGGAGCTGTCCCGACTCCGGTCAGACGACACGGCTGTCTACTATTGTGCTCGGGACTATGGCGATTATGGCATGGACTACTGGGGTCAGGGTACGACTGTAACAGTTAGTAGTGGTGGAGGCGGCAGTGGCGGGGGGGGAAGCGGAGGAGGGGGTTCTGGTGACATAGTTATGACCCAATCCCCAGATAGTTTGGCGGTTTCTCTGGGCGAGAGGGCAACGATTAATTGTCGCGCATCAAAGAGCGTTTCAACGAGCGGATATTCTTTTATGCATTGGTACCAGCAAAAACCCGGACAACCGCCGAAGCTGCTGATCTACTTGGCTTCAAATCTTGAGTCTGGGGTGCCGGACCGATTTTCTGGTAGTGGAAGCGGAACTGACTTTACGCTCACGATCAGTTCACTGCAGGCTGAGGATGTAGCGGTCTATTATTGCCAGCACAGTAGAGAAGTCCCCTGGACCTTCGGTCAAGGCACGAAAGTAGAAATTAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAA 46 CD70 CARMALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASG amino acidYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMT sequenceRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSS (CD70BGGGGSGGGGSGGGGSGDIVMTQSPDSLAVSLGERATINCRASKSVSTSG scFV withYSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQ 41BB)AEDVAVYYCQHSREVPWTFGQGTKVEIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 47 CD70AGATATAGTTATGACCCAATCACCCGATAGTCTTGCGGTAAGCCTGGG scFvGGAGCGAGCAACAATAAACTGTCGGGCATCAAAATCCGTCAGTACA nucleotideAGCGGGTATTCATTCATGCACTGGTATCAACAGAAACCCGGTCAGCC sequenceACCCAAGCTCCTGATTTATCTTGCGTCTAATCTTGAGTCCGGCGTCCCAGACCGGTTTTCCGGCTCCGGGAGCGGCACGGATTTTACTCTTACTATTTCTAGCCTTCAGGCCGAAGATGTGGCGGTATACTACTGCCAGCATTCAAGGGAAGTTCCTTGGACGTTCGGTCAGGGCACGAAAGTGGAAATTAAAGGCGGGGGGGGATCCGGCGGGGGAGGGTCTGGAGGAGGTGGCAGTGGTCAGGTCCAACTGGTGCAGTCCGGGGCAGAGGTAAAAAAACCCGGCGCGTCTGTTAAGGTTTCATGCAAGGCCAGTGGATATACTTTCACCAATTACGGAATGAACTGGGTGAGGCAGGCCCCTGGTCAAGGCCTGAAATGGATGGGATGGATAAACACGTACACCGGTGAACCTACCTATGCCGATGCCTTTAAGGGTCGGGTTACGATGACGAGAGACACCTCCATATCAACAGCCTACATGGAGCTCAGCAGATTGAGGAGTGACGATACGGCAGTCTATTACTGTGCAAGAGACTACGGCGATTATGGCATGGATTACTGGGGCCAGGGCACTACAGTAACCGTTTCCAGC 48 CD70ADIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPP scFv aminoKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREV acidPWTFGQGTKVEIKGGGGSGGGGSGGGGSGQVQLVQSGAEVKKPGASV sequenceKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAF (linkerKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQ underlined) GTTVTVSS 49CD70B CAGGTCCAGTTGGTGCAAAGCGGGGCGGAGGTGAAAAAACCCGGCG scFvCTTCCGTGAAGGTGTCCTGTAAGGCGTCCGGTTATACGTTCACGAAC nucleotideTACGGGATGAATTGGGTTCGCCAAGCGCCGGGGCAGGGACTGAAAT sequenceGGATGGGGTGGATAAATACCTACACCGGCGAACCTACATACGCCGACGCTTTTAAAGGGCGAGTCACTATGACGCGCGATACCAGCATATCCACCGCATACATGGAGCTGTCCCGACTCCGGTCAGACGACACGGCTGTCTACTATTGTGCTCGGGACTATGGCGATTATGGCATGGACTACTGGGGTCAGGGTACGACTGTAACAGTTAGTAGTGGTGGAGGCGGCAGTGGCGGGGGGGGAAGCGGAGGAGGGGGTTCTGGTGACATAGTTATGACCCAATCCCCAGATAGTTTGGCGGTTTCTCTGGGCGAGAGGGCAACGATTAATTGTCGCGCATCAAAGAGCGTTTCAACGAGCGGATATTCTTTTATGCATTGGTACCAGCAAAAACCCGGACAACCGCCGAAGCTGCTGATCTACTTGGCTTCAAATCTTGAGTCTGGGGTGCCGGACCGATTTTCTGGTAGTGGAAGCGGAACTGACTTTACGCTCACGATCAGTTCACTGCAGGCTGAGGATGTAGCGGTCTATTATTGCCAGCACAGTAGAGAAGTCCCCTGGACCTTCGGTCAAGGCACGAAAGTAGAAATTAAA 50 CD70BQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLK scFv aminoWMGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAV acidYYCARDYGDYGMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGDIVMT sequenceQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKWYL (linkerASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFG underlined) QGTKVEIK51 CD70 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGEPTYADAFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSS 52 CD70 VLDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREV PWTFGQGTKVEIK 53Linker GGGGSGGGGSGGGGSG 54 BCMACCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGC rAAVGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGCTCAAGAAGCCCGGAGCCTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCAACACCCTGACCAACTACGTGATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGCTGGAGTGGATGGGCTACATCCTGCCCTACAACGACCTGACCAAGTACAGCCAGAAGTTCCAGGGCAGGGTGACCATCACCAGGGATAAGAGCGCCTCCACCGCCTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGTACTACTGTACAAGGTGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCAGGGCACAACAGTGACCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGGCGGAAGCGAAATCGTGATGACCCAGAGCCCCGCCACACTGAGCGTGAGCCCTGGCGAGAGGGCCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGCACAGCAACGGCAACACCCACCTGCACTGGTACCAGCAGAGACCCGGACAGGCTCCCAGGCTGCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTGCCTGCCAGGTTTAGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCATCAGCAGCGTGGAGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGACCAGCCACATCCCTTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGGTAACCACGTGCGGACCGAGGCTGCAGCGTCGTCCTCCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 55 BCMAGAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTA RHA toAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCA LHAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAGCTCAAGAAGCCCGGAGCCTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCAACACCCTGACCAACTACGTGATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGCTGGAGTGGATGGGCTACATCCTGCCCTACAACGACCTGACCAAGTACAGCCAGAAGTTCCAGGGCAGGGTGACCATCACCAGGGATAAGAGCGCCTCCACCGCCTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGTACTACTGTACAAGGTGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCAGGGCACAACAGTGACCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGGCGGAAGCGAAATCGTGATGACCCAGAGCCCCGCCACACTGAGCGTGAGCCCTGGCGAGAGGGCCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGCACAGCAACGGCAACACCCACCTGCACTGGTACCAGCAGAGACCCGGACAGGCTCCCAGGCTGCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTGCCTGCCAGGTTTAGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCATCAGCAGCGTGGAGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGACCAGCCACATCCCTTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGG 56 BCMAATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCT CARCCACGCAGCAAGGCCGCAGGTGCAGCTGGTGCAGAGCGGAGCCGAG nucleotideCTCAAGAAGCCCGGAGCCTCCGTGAAGGTGAGCTGCAAGGCCAGCG sequenceGCAACACCCTGACCAACTACGTGATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGCTGGAGTGGATGGGCTACATCCTGCCCTACAACGACCTGACCAAGTACAGCCAGAAGTTCCAGGGCAGGGTGACCATCACCAGGGATAAGAGCGCCTCCACCGCCTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGTACTACTGTACAAGGTGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCAGGGCACAACAGTGACCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGGCGGAAGCGAAATCGTGATGACCCAGAGCCCCGCCACACTGAGCGTGAGCCCTGGCGAGAGGGCCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGCACAGCAACGGCAACACCCACCTGCACTGGTACCAGCAGAGACCCGGACAGGCTCCCAGGCTGCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTGCCTGCCAGGTTTAGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCATCAGCAGCGTGGAGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGACCAGCCACATCCCTTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAAAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGA 57 BCMAMALPVTALLLPLALLLHAARPQVQLVQSGAELKKPGASVKVSCKASGN CAR aminoTLTNYVIHWVRQAPGQRLEWMGYILPYNDLTKYSQKFQGRVTITRDKS acidASTAYMELSSLRSEDTAVYYCTRWDWDGFFDPWGQGTTVTVSSGGGG sequenceSGGGGSGGGGSEIVMTQSPATLSVSPGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYSVSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCSQTSHIPYTFGGGTKLEIKSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLSTATKDTYDALHMQALPPR58 BCMA CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGCTCAAGAAGCCCGGAG scFvCCTCCGTGAAGGTGAGCTGCAAGGCCAGCGGCAACACCCTGACCAA nucleotideCTACGTGATCCACTGGGTGAGACAAGCCCCCGGCCAAAGGCTGGAG sequenceTGGATGGGCTACATCCTGCCCTACAACGACCTGACCAAGTACAGCCAGAAGTTCCAGGGCAGGGTGACCATCACCAGGGATAAGAGCGCCTCCACCGCCTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCTGTGTACTACTGTACAAGGTGGGACTGGGACGGCTTCTTTGACCCCTGGGGCCAGGGCACAACAGTGACCGTCAGCAGCGGCGGCGGAGGCAGCGGCGGCGGCGGCAGCGGCGGAGGCGGAAGCGAAATCGTGATGACCCAGAGCCCCGCCACACTGAGCGTGAGCCCTGGCGAGAGGGCCAGCATCTCCTGCAGGGCTAGCCAAAGCCTGGTGCACAGCAACGGCAACACCCACCTGCACTGGTACCAGCAGAGACCCGGACAGGCTCCCAGGCTGCTGATCTACAGCGTGAGCAACAGGTTCTCCGAGGTGCCTGCCAGGTTTAGCGGCAGCGGAAGCGGCACCGACTTTACCCTGACCATCAGCAGCGTGGAGTCCGAGGACTTCGCCGTGTATTACTGCAGCCAGACCAGCCACATCCCTTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAA 59 BCMAQVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEW scFv aminoMGYILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVYY acidCTRWDWDGFFDPWGQGTTVTVSSGGGGSGGGGSGGGGSEIVMTQSPA sequenceTLSVSPGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYSVSN (linkerRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCSQTSHIPYTFGGGTKL underlined) EIK 60BCMA VH QVQLVQSGAELKKPGASVKVSCKASGNTLTNYVIHWVRQAPGQRLEWMGYILPYNDLTKYSQKFQGRVTITRDKSASTAYMELSSLRSEDTAVYY CTRWDWDGFFDPWGQGTTVTVSS61 BCMA VL EIVMTQSPATLSVSPGERASISCRASQSLVHSNGNTHLHWYQQRPGQAPRLLIYSVSNRFSEVPARFSGSGSGTDFTLTISSVESEDFAVYYCSQTSHIP YTFGGGTKLEIK 62CD70 VL RASKSVSTSGYSFMH CDR1 (Kabat) 63 CD70 VL SKSVSTSGYSF CDR1(Chothia) 64 CD70 VL LASNLES CDR2 (Kabat) 65 CD70 VL LAS CDR2 (Chothia)66 CD70 VL QHSREVPWT CDR3 (Kabat) 67 CD70 VL SREVPW CDR3 (Chothia) 68CD70 VH NYGMN CDR1 (Kabat) 69 CD70 VH GYTFTNYGMN CDR1 (Chothia) 70CD70 VH WINTYTGEPTYADAFKG CDR2 (Kabat) 71 CD70 VH NTYTGE CDR2 (Chothia)72 CD70 VH DYGDYGMDY CDR3 (Kabat) 73 CD70 VH CARDYGDYGMDYWG CDR3(Chothia) 74 BCMA VL RASQSLVHSNGNTHLH CDR1 (Kabat) 75 BCMA VLRASQSLVHSNGNTHLH CDR1 (Chothia) 76 BCMA VL SVSNR CDR2 (Kabat) 77 BCMA VLSVSNR CDR2 (Chothia) 78 BCMA VL SQTSHIPYT CDR3 (Kabat) 79 BCMA VLSQTSHIPYT CDR3 (Chothia) 80 BCMA VH NYVIH CDR1 (Kabat) 81 BCMA VHGNTLTNY CDR1 (Chothia) 82 BCMA VH YILPYNDLTKYSQKFQG CDR2 (Kabat) 83BCMA VH LPYNDL CDR2 (Chothia) 84 BCMA VH WDWDGFFDP CDR3 (Kabat) 85BCMA VH WDWDGFFDP CDR3 (Chothia) 86 TRAC AGAGCAACAGTGCTGTGGCC targetsequence 87 anti-CD33 CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCCAR rAAV GTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccactggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaCCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGGAAATCGTCCTCACACAATCCCCGGGGAGCCTCGCAGTCAGTCCTGGGGAACGAGTCACTATGAGCTGCAAATCCAGTCAGAGTGTTTTTTTCTCAAGTAGCCAGAAGAACTACCTCGCATGGTACCAACAAATACCGGGGCAATCTCCCCGCTTGCTTATATACTGGGCAAGTACCCGCGAATCCGGCGTACCGGATCGATTCACGGGATCTGGGTCAGGTACTGATTTCACTTTGACTATCAGCTCTGTTCAGCCTGAAGATTTGGCAATTTACTACTGTCACCAATACTTGAGTAGCCGAACTTTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAGGGGGAGGTTCTGGTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACAGTTGCAACAGCCAGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAGATGAGCTGTAAAGCAAGTGGATACACCTTCACCTCCTACTATATACATTGGATTAAGCAAACTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATACCCCGGGAACGATGATATATCATACAACCAAAAATTTCAAGGCAAGGCGACTCTGACTGCCGATAAGAGTAGCACAACAGCTTACATGCAGCTTTCTTCCCTGACCAGCGAAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCGCCTGCGATACTTTGATGTCTGGGGTCAAGGAACTACAGTTACTGTATCAAGCAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGGTAACCACGTGCGGACCGAGGCTGCAGCGTCGTCCTCCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 88 signalMLLLVTSLLLCELPHPAFLLIP peptide 89 signal MALPVTALLLPLALLLHAARP peptide90 CD8a FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG trans-AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR membrane domain 91 CD70UCACCAAGCCCGCGACCAAU sgRNA (E1_T1) spacer 92 CD70 AUCACCAAGCCCGCGACCAAsgRNA (E1_T3) spacer 93 CD70 CGGUGCGGCGCAGGCCCUAU sgRNA (E1_T4) spacer94 CD70 GCUUUGGUCCCAUUGGUCGC sgRNA (E1_T7) spacer 95 CD70GCCCGCAGGACGCACCCAUA sgRNA (E1_T8) spacer 96 CD70 GUGCAUCCAGCGCUUCGCACsgRNA (E1_T10) spacer 97 CD70 CAGCUACGUAUCCAUCGUGA sgRNA (E3_T1) spacer98 TRAC AGAGCAACAGUGCUGUGGCC spacer 99 β2M GCUACUCUCUCUUUCUGGCC sgRNAspacer 100 PD-1 CUGCAGCUUCUCCAACACAU sgRNA spacer 101 CD70U*C*A*CCAAGCCCGCGACCAAU sgRNA (E1_T3) spacer 102 CD70A*U*C*ACCAAGCCCGCGACCAA sgRNA (E1_T4) spacer 103 CD70C*G*G*UGCGGCGCAGGCCCUAU sgRNA (E1_T7) spacer 104 CD70G*C*U*UUGGUCCCAUUGGUCGC sgRNA (E1_T8) spacer 105 CD70G*C*C*CGCAGGACGCACCCAUA sgRNA (E1_T10) spacer 106 CD70G*U*G*CAUCCAGCGCUUCGCAC sgRNA (E3_T10) spacer 107 CD70C*A*G*CUACGUAUCCAUCGUGA sgRNA (E1_T3) spacer 108 TRACA*G*A*GCAACAGUGCUGUGGCC spacer 109 β2M G*C*U*ACUCUCUCUUUCUGGCC sgRNAspacer 110 PD-1 C*U*G*CAGCUUCUCCAACACAU sgRNA spacer 111 CD70TCACCAAGCCCGCGACCAATGGG sgRNA (E1_T1) with PAM 112 CD70ATCACCAAGCCCGCGACCAATGG sgRNA (E1_T3) with PAM 113 CD70CGGTGCGGCGCAGGCCCTATGGG sgRNA (E1_T4) with PAM 114 CD70GCTTTGGTCCCATTGGTCGCGGG sgRNA (E1_T7) with PAM 115 CD70GCCCGCAGGACGCACCCATAGGG sgRNA (E1_T8) with PAM 116 CD70GTGCATCCAGCGCTTCGCACAGG sgRNA (E1_T10) with PAM 117 CD70CAGCTACGTATCCATCGTGATGG sgRNA (E3_T1) with PAM 118 TRACAGAGCAACAGTGCTGTGGCCTGG sgRNA with PAM 119 β2M GCTACTCTCTCTTTCTGGCCTGGsgRNA with PAM 120 PD-1 CTGCAGCTTCTCCAACACATCGG sgRNA with PAM 121 CD28TCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCC nucleotideTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCC sequenceCCACGAGACTTCGCTGCGTACAGGTCC 122 TRAC-GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTA LHAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTT CA 123 EF1αGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCC promoterCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTC AGGTGTCGTGA 124Synthetic AATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTG poly(A) TGsignal 125 TRAC- TGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACA RHAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTC AATGAGAAAGG 126 CD8aIYIWAPLAGTCGVLLLSLVITLY trans- membrane 127 CD70TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGcccaacttttccatctcaactca forwardccccaagtg primer 128 CD70GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGcccctcctgcgctagcgga reverse primer 129CD70 Indel CACACCACGAGGCAGATCACCAAGCCCGCG- CAATGGGACCAAAGCAGCCCGCAGGACG130 CD70 Indel CACACCACGAGGCAGATCACCAAGCCCGCGAACCAATGGGACCAAAGCAGCCCGCAGGACG 131 CD70 Indel CACACCACGAGGCAGATC------------ACCAATGGGACCAAAGCAGCCCGCAGGACG 132 CD70 IndelCACACCACGAGGCAGATCACCAAGCCCGCG- CCAATGGGACCAAAGCAGCCCGCAGGACG 133CD70 Indel CACACCACGAGGCAGATCACCAAGCCCGC- ACCAATGGGACCAAAGCAGCCCGCAGGACG134 CD70 Indel CACACCACGAGGCAGATCACCA-------------------------AGCCCGCAGGACG 135 Anti-CD33GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGT CARAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTC DonorAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGAT LHA toTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATG RHACCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGT 41BBTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGT costim.TATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccactggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaCCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTGTTGCTCCACGCAGCAAGGCCGGAAATCGTCCTCACACAATCCCCGGGGAGCCTCGCAGTCAGTCCTGGGGAACGAGTCACTATGAGCTGCAAATCCAGTCAGAGTGTTTTTTTCTCAAGTAGCCAGAAGAACTACCTCGCATGGTACCAACAAATACCGGGGCAATCTCCCCGCTTGCTTATATACTGGGCAAUACCCGCGAATCCGGCGTACCGGATCGATTCACGGGATCTGGGTCAGGTACTGATTTCACTTTGACTATCAGCTCTGTTCAGCCTGAAGATTTGGCAATTTACTACTGTCACCAATACTTGAGTAGCCGAACTTTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAGGGGGAGGTTCTGGTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACAGTTGCAACAGCCAGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAGATGAGCTGTAAAGCAAGTGGATACACCTTCACCTCCTACTATATACATTGGATTAACCAAACTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATACCCCGGGAACGATGATATATCATACAACCAAAAATTTCAAGGCAAGGCGACTCTGACTGCCGATAAGAGTAGCACAACAGCTTACATGCAGCTTTCT1CCCTGACCAGCGAAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCGCCTGCGATACTTTGATGTCTGGGGTCAAGGAACTACAGTTACT(TATCAAGCAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCAlCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCCCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCG(AGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAA1AAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGC1TTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGICAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGACCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCT CAATGAGAAAGG 136 Anti-CD33CCACCATGGCGCTTCCGGTGACAGCACTGCTCCTCCCCTTGGCGCTG CARTTGCTCCACGCAGCAAGGCCGGAAATCGTCCTCACACAATCCCCGG 41BBGGAGCCTCGCAGTCAGTCCTGGGGAACGAGTCACTATGAGCTGCAA costimATCCAGTCAGAGTGTTTTTTTCTCAAGTAGCCAGAAGAACTACCTCGCATGGTACCAACAAATACCGGGGCAATCTCCCCGCTTGCTTATATACTGGGCAAGTACCCGCGAATCCGGCGTACCGGATCGATTCACGGGATCTGGGTCAGGTACTGATTTCACTTTGACTATCAGCTCTGTTCAGCCTGAAGATTTGGCAATTTACTACTGTCACCAATACTTGAGTAGCCGAACTTTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAGGGGGAGGTTCTGGTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACAGTTGCAACAGCCAGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAGATGAGCTGTAAAGCAAGTGGATACACCTTCACCTCCTACTATATACATTGGATTAAGCAAACTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATACCCCGGGAACGATGATATATCATACAACCAAAAATTTCAAGGCAAGGCGACTCTGACTGCCGATAAGAGTAGCACAACAGCTTACATGCAGCTTTCTTCCCTGACCAGCGAAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCGCCTGCGATACTTTGATGTCTGGGGTCAAGGAACTACAGTTACTGTATCAAGCAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTT GGTTTTTTGTGTG 137Anti-CD33 EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQS scFvPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQYLS LinkerSRTFGQGTKLEIKGGGGGSGGGGSGGGGSQVQLQQPGAEVVKPGASV underlinedKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTT VTVSS 138 Anti-CD33GAAATCGTCCTCACACAATCCCCGGGGAGCCTCGCAGTCAGTCCTG scFvGGGAACGAGTCACTATGAGCTGCAAATCCAGTCAGAGTGTTTTTTTCTCAAGTAGCCAGAAGAACTACCTCGCATGGTACCAACAAATACCGGGGCAATCTCCCCGCTTGCTTATATACTGGGCAAGTACCCGCGAATCCGGCGTACCGGATCGATTCACGGGATCTGGGTCAGGTACTGATTTCACTTTGACTATCAGCTCTGTTCAGCCTGAAGATTTGGCAATTTACTACTGTCACCAATACTTGAGTAGCCGAACTTTCGGCCAGGGCACGAAGCTCGAAATCAAGGGCGGAGGGGGAGGTTCTGGTGGGGGCGGTTCTGGCGGTGGAGGAAGCCAAGTACAGTTGCAACAGCCAGGGGCGGAGGTCGTAAAACCTGGGGCGTCTGTCAAGATGAGCTGTAAAGCAAGTGGATACACCTTCACCTCCTACTATATACATTGGATTAAGCAAACTCCGGGTCAGGGGCTGGAATGGGTTGGCGTTATATACCCCGGGAACGATGATATATCATACAACCAAAAATTTCAAGGCAAGGCGACTCTGACTGCCGATAAGAGTAGCACAACAGCTTACATGCAGCTTTCTTCCCTGACCAGCGAAGATTCAGCAGTTTACTACTGCGCTCGGGAAGTGCGCCTGCGATACTTTGATGTCTGGGGTCAAGGAACTACAGTTACTGTATCAAGC 139 Anti-CD33MALPVTALLLPLALLLHAARPEIVLTQSPGSLAVSPGERVTMSCKSSQS CARVFFSSSQKNYLAWYQQIPGQSPRLLIYWASTRESGVPDRFTGSGSGTDF 41BBTLTISSVQPEDLAIYYCHQYLSSRTFGQGTKLEIKGGGGGSGGGGSGGG costim.GSQVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGTTVTVSSSAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 140 anti-CD33 QVQLQQPGAEVVKPGASVKMSCKASGYTFTSYYIH WIKQTPGQGLEW antibody VG VIYPGNDDISYNQKFQGKATLTADKSSTTAYMQLSSLTSEDSAVYY VH CAR EVRLRYFDV WGQGTTVTVSS CDRsunderlined and in bold 141 anti-CD33 EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLA WYQQIPGQS antibody PRLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYC H Q YLS VL SRT FGQGTKLEIK CDRsunderlined and in bold 142 anti-CD33 SYYIH antibody VH CDR1 (Kabat) 143anti-CD33 VIYPGNDDISYNQKFQG antibody ' VH CDR2 (Kabat) 144 anti-CD33EVRLRYFDV antibody VH CDR3 (Kabat) 145 anti-CD33 KSSQSVFFSSSQKNYLAantibody VL CDR1 (Kabat & Chothia) 146 anti-CD33 WASTRES antibodyVL CDR2 (Kabat & Chothia 147 anti-CD33 HQYLSSRT antibody VL CDR3(Kabat & Chothia) 148 Anti-CD19ATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCCA CARGCGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCACCAGTAG CD8[tm]-CTTGTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAA CD28[co-GTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGA stimulatoryCGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCG domain]-GAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCC CD3z)TTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGA 149 Anti-CD19MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDI CARSKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNL CD8[tm]-EQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEV CD28[co-KLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI stimulatoryWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY domain]-YYGGSYAMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAPTI CD3z)ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLV Amino AcidITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR 150Anti-CD19 GATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGG scFvAGACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGT 151 CD19 scFvDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY amino acidHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG sequenceGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTV LinkerSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDN underlinedSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 152 Anti-CD19EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLG VHVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKH YYYGGSYAMDYWGQGTSVTVSS153 Anti-CD19 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY VLHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG GGTKLEIT 154Anti-CD19 GSTSGSGKPGSGEGSTKG scFv linker 155 anti-CD19CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGC CAR rAAVGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCCAGCGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGGTAACCACGTGCGGACCGAGGCTGCAGCGTCGTCCTCCCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCTGCCTGCAGG 156Anti-CD19 GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTA CAR LHAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCA to RHAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCCAGCGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCGCTATATATTATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACTGCATATGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGG AGCTCAATGAGAAAGG 157 CD70TCACCAAGCCCGCGACCAAT sgRNA (E1_T1) 158 CD70 ATCACCAAGCCCGCGACCAA sgRNA(E1_T3) 159 CD70 CGGTGCGGCGCAGGCCCTAT sgRNA (E1_T4) 160 CD70GCTTTGGTCCCATTGGTCGC sgRNA (E1_T7) 161 CD70 GCCCGCAGGACGCACCCATA sgRNA(E1_T8) 162 CD70 GTGCATCCAGCGCTTCGCAC sgRNA (E1_T10) 163 CD70CAGCTACGTATCCATCGTGA sgRNA (E3_T1) 164 β2M sgRNA GCTACTCTCTCTTTCTGGCC165 PD-1 sgRNA CTGCAGCTTCTCCAACACAT 166 anti-CD19 RASQDISKYLN VL CDR1(Kabat) 167 anti-CD19 HTSRLHS VL CDR2 (Kabat) 168 anti-CD19 QQGNTLPYTVL CDR3 (Kabat) 169 anti-CD19 DYGVS VH CDR1 (Kabat) 170 anti-CD19VIWGSETTYYNSALKS VH CDR2 (Kabat) 171 anti-CD19 HYYYGGSYAMDY VH CDR3(Kabat) 172 anti-CD19 RASQDISKYLN VL CDR1 (Chothia) 173 anti-CD19HTSRLHS VL CDR2 (Chothia) 174 anti-CD19 QQGNTLPYT VL CDR3 (Chothia) 175anti-CD19 GVSLPDY VH CDR1 (Chothia) 176 anti-CD19 WGSET VH CDR2(Chothia) 177 anti-CD19 HYYYGGSYAMDY VH CDR3 (Chothia) 178 anti-CD33GYTFTSY VH CDR1 (Chothia) 179 anti-CD33 YPGNDD VH CDR2 (Chothia) 180anti-CD33 EVRLRYFDV VH CDR3 (Chothia)

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The terms “about” and “substantially” preceding a numerical value mean±10% of the recited numerical value.

Where a range of values is provided, each value between the upper andlower ends of the range are specifically contemplated and describedherein.

1. An engineered T cell comprising a disrupted CD70 gene and a nucleicacid encoding a chimeric antigen receptor (CAR) that does not bind CD70.2. The engineered T cell of claim 1, further comprising a disrupted Tcell receptor alpha constant region (TRAC) gene, a disruptedbeta-2-microglobulin (β2M) gene, or both.
 3. (canceled)
 4. Theengineered T cell of claim 2, wherein the disrupted TRAC gene comprisesthe nucleic acid encoding the CAR.
 5. The engineered T cell of claim 1,wherein the CAR comprises an ectodomain that binds an antigen selectedfrom the group consisting of B cell maturation antigen (BCMA), CD33, andCD19.
 6. The engineered T cell of claim 5, wherein the ectodomaincomprises an anti-BCMA antibody, an anti-CD33 antibody, or an anti-CD19antibody.
 7. The engineered T cell of claim 5, wherein the ectodomaincomprises an anti-BCMA single-chain variable fragment (scFv), ananti-CD33 scFv, or an anti-CD19 scFv.
 8. The engineered T cell of claim7, wherein the ectodomain comprises an anti-BCMA scFv, which comprisesthe same variable heavy (VH) chain complementarity determining regions(CDRs) and the same variable light (VL) chain CDRs as a referenceantibody, and wherein the reference antibody comprises a VH set forth asSEQ ID NO: 60 and a VL set forth as SEQ ID NO:
 61. 9. The engineered Tcell of claim 8, wherein the anti-BCMA scFv comprises VH and VL chainscomprising the amino acid sequences set forth in SEQ ID NOs: 60 and 61,respectively.
 10. The engineered T cell of claim 8, wherein theanti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 59.11-13. (canceled)
 14. The engineered T cell of claim 7, wherein theectodomain comprises an anti-CD33 scFv, which comprises the same VH CDRsand the same VL chain CDRs as a reference antibody, and wherein thereference antibody comprises a VH set forth as SEQ ID NO: 140 and a VLset forth as SEQ ID NO:
 141. 15. The engineered T cell of claim 14,wherein the anti-CD33 scFv comprises VH and VL chains comprising theamino acid sequences set forth in SEQ ID NOs: 140 and 141, respectively.16. The engineered T cell of claim 14, wherein the anti-CD33 scFvcomprises the amino acid sequence of SEQ ID NO:
 137. 17-19. (canceled)20. The engineered T cell of claim 19, wherein the ectodomain comprisesan anti-CD19 scFv, which comprises the same VH CDRs and the same VLchain CDRs as a reference antibody, and wherein the reference antibodycomprises a VH set forth as SEQ ID NO: 152 and a VL set forth as SEQ IDNO:
 153. 21. The engineered T cell of claim 20, wherein the anti-CD19scFv comprises VH and VL chains comprising the amino acid sequences setforth in SEQ ID NOs: 152 and 153, respectively.
 22. The engineered Tcell of claim 20, wherein the anti-CD19 scFv comprises the amino acidsequence of SEQ ID NO:
 151. 23-42. (canceled)
 43. The engineered T cellof claim 1, wherein the engineered T cell further comprises a disruptedprogrammed cell death-1 (PD-1) gene.
 44. The engineered T cell of claim1, wherein the engineered T cell maintains cytotoxicity following 5rechallenges or following 10 rechallenges with a target cell, whereinthe target cell expresses an antigen specific for the CAR. 45.(canceled)
 46. The engineered T cell of claim 44, wherein the targetcell is a cancer cell.
 47. A population of cells comprising engineered Tcells, wherein the engineered T cells comprise a disrupted CD70 gene anda nucleic acid encoding a CAR that does not bind CD70.
 48. Thepopulation of cells of claim 47, further comprising a disrupted TRACgene, a disrupted β2M gene, or both.
 49. (canceled)
 50. The populationof cells of claim 48, wherein the disrupted TRAC gene comprises thenucleic acid encoding the CAR.
 51. The population of cells of claim 47,wherein the CAR comprises an ectodomain that binds an antigen selectedfrom the group consisting of B cell maturation antigen (BCMA), CD33, andCD19.
 52. The population of cells of claim 51, wherein the ectodomaincomprises an anti-BCMA antibody, an anti-CD33 antibody, or an anti-CD19antibody.
 53. The population of cells of claim 51, wherein theectodomain comprises an anti-BCMA single-chain variable fragment (scFv),an anti-CD33 scFv, or an anti-CD19 scFv.
 54. The population of cells ofclaim 53, wherein the ectodomain comprises an anti-BCMA scFv, whichcomprises the same variable heavy (VH) chain complementarity determiningregions (CDRs) and the same variable light (VL) chain CDRs as areference antibody, wherein the reference antibody comprises a VH setforth as SEQ ID NO: 60 and a VL set forth as SEQ ID NO:
 61. 55. Thepopulation of cells of claim 54, wherein the anti-BCMA scFv comprises VHand VL chains comprising the amino acid sequences set forth in SEQ IDNOs: 60 and 61, respectively.
 56. The population of cells of claim 54,wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ IDNO:
 59. 57-59. (canceled)
 60. The population of cells of claim 53,wherein the ectodomain comprises an anti-CD33 scFv, which comprises thesame VH CDRs and the same VL chain CDRs as a reference antibody, whereinthe reference antibody comprises a VH set forth as SEQ ID NO: 140 and aVL set forth as SEQ ID NO:
 141. 61. The population of cells of claim 60,wherein the anti-CD33 scFv comprises VH and VL chains comprising theamino acid sequences set forth in SEQ ID NOs: 140 and 141, respectively.62. The population of cells of claim 60, wherein the anti-CD33 scFvcomprises the amino acid sequence of SEQ ID NO:
 137. 63-65. (canceled)66. The population of cells of claim 53, wherein the ectodomaincomprises an anti-CD19 scFv, which comprises the same VH CDRs and thesame VL chain CDRs as a reference antibody, wherein the referenceantibody comprises a VH set forth as SEQ ID NO: 152 and a VL set forthas SEQ ID NO:
 153. 67. The population of cells of claim 66, wherein theanti-CD19 scFv comprises VH and VL chains comprising the amino acidsequences set forth in SEQ ID NOs: 152 and 153, respectively.
 68. Thepopulation of cells of claim 66, wherein the anti-CD19 scFv comprisesthe amino acid sequence of SEQ ID NO:
 151. 69-80. (canceled)
 81. Thepopulation of cells of claim 48, wherein there is a deletion in the TRACgene relative to unmodified T cells.
 82. The engineered T cell of claim81, wherein the deletion is 15-30 base pairs.
 83. The engineered T cellof claim 81, wherein the deletion is 20 base pairs.
 84. The engineered Tcell of claim 81, wherein the deletion comprises SEQ ID NO:
 86. 85-89.(canceled)
 90. The population of cells of claim 47, wherein theengineered T cell maintains cytotoxicity following 5 rechallenges orfollowing 10 rechallenges with a target cell, wherein the target cellexpresses an antigen specific for the CAR.
 91. (canceled)
 92. Thepopulation of cells of claim 90, wherein the target cell is a cancercell.
 93. The population of cells of claim 48, wherein the disrupted β2Mgene comprises at least one nucleotide sequence selected from any one ofSEQ ID NOS: 9-14.
 94. The population of cells of claim 47, wherein thedisrupted CD70 gene comprises at least one nucleotide sequence selectedfrom any one of SEQ ID NOS: 129-134.
 95. The population of cells ofclaim 48, wherein at least 90% of the engineered T cells do not expressa detectable level of TCR surface protein.
 96. The population of cellsof claim 47, wherein the engineered T cells: (a) exhibit increasedcellular proliferative capacity; (b) exhibit increased cell lysis; (c)exhibit reduced cellular exhaustion; (d) maintain cytokine-dependentproliferation; (e) exhibit increased cytokine secretion; or (f) anycombination of (a)-(e), relative to control T cells, wherein control Tcells express endogenous CD70 protein.
 97. A method comprisingadministering to a subject the population of cells of claim
 47. 98. Themethod of claim 97, wherein the engineered T cells are engineered humanT cells.
 99. The method of claim 97, wherein the subject has a cancer.100. The method of claim 99, wherein the cancer expresses BMCA, CD19,CD33 or combinations thereof.
 101. (canceled)
 102. The method of claim99, wherein the cancer is a solid tumor malignancy or a hematologicalmalignancy.
 103. The method of claim 102, wherein the solid tumormalignancy is selected from the group consisting of: ovarian tumor,pancreatic tumor, kidney tumor, lung tumor, and intestinal tumor.104-110. (canceled)
 111. A method for producing an engineered T cell,the method comprising: (a) delivering to a T cell an RNA-guidednuclease, a gRNA targeting a CD70 gene, and a vector comprising a donortemplate that comprises a nucleic acid encoding a CAR; and (b) producingan engineered T cell comprising a disrupted CD70 gene and expressing theCAR. 112-114. (canceled)
 115. A method for producing an engineered Tcell, the method comprising (a) delivering to a T cell an RNA-guidednuclease, a gRNA targeting a TRAC gene, a gRNA targeting a β2M gene, agRNA targeting a CD70 gene, and a vector comprising a donor templatethat comprises a nucleic acid encoding a CAR; and (b) producing anengineered T cell. 116-121. (canceled)
 122. A method for producing anengineered T cell for immunotherapy against a target cell, comprising:(a) disrupting a CD70 gene in a T cell, and (b) expressing a CAR thatbinds to an antigen expressed on the target cell, wherein the antigen isnot CD70. 123-156. (canceled)
 157. A method of increasing proliferationor reducing exhaustion of T cells, comprising disrupting the CD70 genein the T cells. 158-161. (canceled)
 162. The engineered T cell of claim5, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 57,SEQ ID NO:139, or SEQ ID NO:149. 163-167. (canceled)
 168. The populationof cells of claim 51, wherein the CAR comprises the amino acid sequenceof SEQ ID NO: 57, SEQ ID NO:139, or SEQ ID NO:149. 169-181. (canceled)